La raison d'être of in situ electro-mobilization, phyto ... · TPB DNA rRNA NADPH tRNA df PEP Ini...

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La raison d'être of in situ electro-mobilization, phyto-extraction and phyto- stabilization of lithium tailing in heterogeneous rhizosphere by Brassica juncea and the monocotyledonous plants Zohrab Keropian A Thesis In The Department of Building, Civil and Environmental Engineering Presented in Partial Fulfillment of the Requirements for the Degree of Master of Applied Sciences (Civil) at Concordia University Montreal, Quebec, Canada August 2012 © Zohrab Keropian, 2012

Transcript of La raison d'être of in situ electro-mobilization, phyto ... · TPB DNA rRNA NADPH tRNA df PEP Ini...

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La raison d'être of in situ electro-mobilization, phyto-extraction and phyto-

stabilization of lithium tailing in heterogeneous rhizosphere by Brassica

juncea and the monocotyledonous plants

Zohrab Keropian

A Thesis

In

The Department of Building, Civil and Environmental Engineering

Presented in Partial Fulfillment of the Requirements for the

Degree of Master of Applied Sciences (Civil) at Concordia University

Montreal, Quebec, Canada

August 2012

© Zohrab Keropian, 2012

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CONCORDIA UNIVERSITY

School of Graduate Studies

This is to certify that the thesis prepared

By: Zohrab Keropian

Entitled: La raison d'être of in situ electro-mobilization, phyto-extraction and phyto- stabilization of

lithium tailing in heterogeneous rhizosphere by Brassica juncea and the monocotyledonous plants

and submitted in partial fulfillment of the requirements for the degree of

Master of Applied Sciences (Civil) at the Department of Building, Civil and Environmental

Engineering

complies with the regulations of the University and meets the accepted standards with respect

to originality and quality.

Signed by the final Examining Committee:

_______________Dr. A.M. Hanna_____________ Chair

_______________Dr. S. Williamson___________ Examiner

_______________Dr. S. Rahaman_____________ Examiner

_______________Dr. M. Elektorowicz__________ Supervisor

Approved by ____________________________________________________

Chair of Department or Graduate Program Director

_____________ 2012 __________________________

Dean of Faculty

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Abstract

The research tested the Brassica juncea ability, to phytoextract and phytostabilize lithium from mine

tailings in lieu with vanadium and chromium, sown in a heterogeneous acidic rhizosphere. Five

different heterogeneous growth media formulations were prepared from lithium mine tailings,

homogenized peat and dewatered municipal biosolids. The Brassica juncea was grown for eighty six

days, under homogeneous growing conditions, irrigated bi-daily with organic fertilizer, amended with

LiCl, harvested and chemically analyzed. The phytoextraction and phytostabilization data revealed that

the Brassica juncea was capable of absorbing more vanadium in its physiological parts rather than

lithium and chromium. Likewise the monocotyledonous plant was grown homogeneously on the most

favorable growth media, amended with lithium chloride and was able to phytoharness and

phytostabilize more lithium rather than chromium per dry weight basis. In botanical efficiency

parameters the monocotyledonous plant was ten times more efficient than the Brassica juncea in the

bioaccumulation and efficiency removal rates for lithium and twice as much as for chromium. The

relative growth rate of the monocotyledonous plant was twice as much as the Brassica juncea.

Moreover, it surpassed the monocotyledonous plant in translocation indexes for chromium more than

six times and twenty times for lithium. The findings revealed the possibility of a three way symbiosis

formed between the hyperaccumulant plant grown in a heterogeneous rhizosphere and coupled with

EK system at certain growth periods that will result in an increased electromigration and

electrophoresis of heavy metals in the growth media solution.

Keywords: Brassica juncea, monocotyledonous plant, lithium, vanadium, chromium, phytoharness

phytostabilization, phytoextraction, growth media, organic fertilizer, peat, biosolids, lithium

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mine tailing, relative growth rate, bioaccumulation ratio, translocation index, efficiency of

removal, electrokinetics, electromigration, electrophoresis.

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Acknowledgement

First and foremost, I would like to thank my academic advisor and mentor at Concordia University, the

Department of Building, Civil and Environmental Engineering, Prof. Maria Elektorowicz. This thesis

could not have been written without who not only served as my supervisor but also encouraged and

challenged me throughout my academic program, never accepting less than my upmost efforts. In

addition, this study was supported graciously by the NSERC Discovery Grant awarded to Dr

Elektorowicz. I owe a sincere and earnest thankfulness to Prof. Dallas Kessler, Chady Stephan, Claude

Devreau, Ritch Nally and the “Agriculture and Agri-Food Canada” research branch in Saskatoon, SK. for

the provision of the Brassica juncea Czern var. Cutlass accession CN: 46238 seeds.

Finally, I would like to dedicate my thesis to my parents and especially to my Mom for her endless

love and dedication towards my graduate studies. It is a great pleasure to express my heartfelt

gratitude to everyone who helped me achieve my goals specially to the members of my ‘special forces’

team including my brother Dr. Chant and my two lovely sisters Ani and Jasmine.

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LIST OF TABLES

Table 2.1: EC ₍₁₋₅₎ to EC ₍e₎ conversion factors...…………………………………………………………..…................58

Table 2.2: Biosolids contents............................……………………………………………………………..................60

Table 2.3: ORP, pH, salinity and TDS characteristics in per group basis...........………......…....….........64

Table 2.4: Metal mass content in P, de-B, LMT and organic fertilizer...............................................72

Table 2.5: Initial metal content in five different GM...........................................................…...........75

Table 3.1: Leachate characteristics per group and wash basis..........................................................85

Table 3.2: Leachate metal mass per Group and wash basis..............................................................88

Table 3.3: Metal mass left in Group after CWP.................................................................................90

Table 3.4: pH, ORP and EC in five different groups.........................................................................105

Table 3.5: Metal mass content in five different Groups..................................................................108

Table 3.6: Metal mass difference between column and EK leachate..............................................110

Table 3.7: Moisture content difference per slice and group basis..................................................112

Table 4.1: Void volume and porosity per group basis.....................................................................116

Table 4.2: Metal mass content in Brassica juncea physiological parts and

rhizosphere........................................................................................................................124

Table 4.3: Remaining metal mass content after the phytoextraction ............................................128

Table 4.4: Brassica juncea efficiency parameters............................................................................132

Table 4.5: Metal mass content in monocotyledonous plant...........................................................142

Table 4.6: Monocotyledonous plant efficiency parameters............................................................143

Table 5.1: LMT texture data in detail...............................................................................................152

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LIST OF FIGURES

Figure 2.1: Methodological flow chart approach..............………………………………….....……..................51

Figure 2.2: Lithium mine tailing..………………………………………………………………………….......................…55

Figure 2.3: LMT cumulative grading curve...………………….……………………………………………................…56

Figure 2.4: Homogenized peat sample..............................................................................................59

Figure 2.5: A fresh Biosolids sample..................................................................................................61

Figure 2.6: Metal mass in a) peat, b) de-B, c) LMT and d) organic fertilizer...……............................73

Figure 3.1: pH vs. ORP vs. EC per group and wash basis...................................................................86

Figure 3.2: Variation of metal mass content per Group and wash basis...........................................89

Figure 3.3: Metal mass left in Groups after column washing procedure..........................................91

Figure 3.4: Initial metal mass content difference..............................................................................92

Figure 3.5: Metal mass removed.......................................................................................................93

Figure 3.6: Initial vs. left vs. leachate metal mass content per group basis......................................94

Figure 3.7: Column apparatus...........................................................................................................98

Figure 3.8: Cap of the EK cell and the stainless steel rods...............................................................101

Figure 3.9: Bottom plate of the EK cell in an upright position.........................................................102

Figure 3.10: EK setup and the DC power generator in mesh...........................................................103

Figure 3.11: pH, ORP and EC per group basis..................................................................................107

Figure 3.12: Metal mass difference between EK vs. column leachate............................................111

Figure 3.13: Moisture content difference in EK cell per group basis...............................................112

Figure 4.1: Woods TIM 1000™ timer...…………………………………………..……………………........................115

Figure 4.2: Organic fertilizer.……………………………………………………………………..……………......…...........115

Figure 4.3: Chronology of germination Brassica juncea growth in Group 4....................................117

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Figure 4.4: Metal mass content in Brassica juncea physiological parts...........................................125

Figure 4.5: Li and Cr phytoextraction and phytostabilization in different Brassica juncea

physiological parts...........................................................................................................126

Figure 4.6: Vanadium phytoextraction and phytostabilization in different Brassica juncea

physiological parts...........................................................................................................127

Figure 4.7: Comparison between prior and after phytoextraction of metallic element mass

content............................................................................................................................129

Figure 4.8: Groups 1 and 5 growth media formulations..................................................................133

Figure 4.9: Monocotyledonous plant growth and harvest..............................................................140

Figure 4.10: Li and Cr phytoextraction and phytostabilization in monocotyledonous

plant................................................................................................................................144

Figure 4.11: Brassica juncea vs. monocotyledonous plant accumulation of Li and

Cr.....................................................................................................................................145

Figure 4.12: Brassica juncea and monocotyledonous plant in botanical efficiency terms for Li and

Cr.....................................................................................................................................146

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The Acronyms

The Acronyms The complete terminology

Temp

Det of M

Det of OM

AAS

ORP

TPB

DNA

rRNA

NADPH

tRNA

df

PEP

Ini

Lea Or L

GM

Homo

Di

LMT

A

EK

DC

Temperature

Determination of moisture

Determination of organic matter

Atomic absorption spectrometer

Oxidation reduction potential

Tailing Peat Biosolids

Deoxyribonucleic acid,

ribosomal RNA,

Nicotinamide adenine dinucleotide

Transfer RNA.

Dilution factor.

Phospho-enol-pyruvate.

Initial

Leachate,

Growth Media.

Homogenous

Digestion

Lithium mine tailing.

Amperage.

Electrokinetics.

Direct Current.

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V

Ref

DW

SD

CL

AL

M-GM

A-GM

C-GM

MC

DW

N/S

ppb

ppm

De-B

CWP

E of R

BAR

RGR

TI

AMD

PPE

EK-GM

Volts.

Reference.

Dry Weight.

Standard Deviation.

Cathode Leachate.

Anode Leachate.

Middle Growth Media.

Anode Growth Media.

Cathode Growth Media.

Monocotyledonous.

Dry Weight.

Nitrogen to sulphur ratio.

Parts per billion.

Parts per million.

Dewatered biosolids.

Column wash procedure

Efficiency of removal

Bioaccumulation ratio

Relative growth rate

Translocation index

Acid mine drainage

Polypropylene

Elektrokinetics-Growth Media

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Table of Contents

Chapter One: Statement of the problem....................................................................................................1

Objectives...............................................................................................................................................3

Detailed objectives................................................................................................................................3

Phase One: Literature review..................................................................................................................4

1.1.1) Introduction to phytoremediation...........................................................................................4

1.1.2) Prerequisites of successful ecological restoration...................................................................8

1.1.3) Brassica juncea “hyperaccumulant plant”...............................................................................9

1.1.3.1 General description.............................................................................................................9

1.1.3.2- Origin and cultivation......................................................................................................10

1.1.3.3- Harvesting........................................................................................................................12

1.1.3.4- Benefits............................................................................................................................12

1.1.3.5- Diseases...........................................................................................................................13

1.1.3.6- Folk medicine...................................................................................................................13

1.1.3.7- Chemical constituents.....................................................................................................14

Phase Two: Mechanisms of mineral uptake and storage in hyperaccumulator plant.........................16

1.2.1) Phyto-uptake and accumulation............................................................................................16

1.2.2) Toxic metal resistance mechanism.........................................................................................17

1.2.2.1- Chelating process.............................................................................................................18

1.2.2.2- Biotransformation and compartmentalization process...................................................18

1.2.2.3- Cellular repair mechanism...............................................................................................19

1.2.2.4- Root and shoot uptake and accumulation.......................................................................20

1.2.2.5- Phytovolatilization...........................................................................................................21

Phase Three: Overview of metal in lithium mine tailing.....................................................................22

1.3.0) Geological formation...............................................................................................................22

1.3.1) Chromium...............................................................................................................................23

1.3.1.1- Introduction....................................................................................................................23

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1.3.1.2- Chromium as a contaminant...........................................................................................23

1.3.1.3- Phytoextraction of chromium.........................................................................................24

1.3.2) Vanadium...............................................................................................................................26

1.3.2.1- Introduction.....................................................................................................................26

1.3.2.2- Vanadium as contaminant...............................................................................................27

1.3.2.3- Phytoextraction of vanadium..........................................................................................29

1.3.3) Iron.........................................................................................................................................30

1.3.3.1- Mineral and availability...................................................................................................30

1.3.3.2- Iron in plant physiology...................................................................................................31

1.3.4) Potassium................................................................................................................................32

1.3.4.1- Mineral and availability...................................................................................................32

1.3.4.2- Potassium in plant physiology.........................................................................................33

1.3.5) Calcium....................................................................................................................................35

1.3.5.1- Mineral and availability...................................................................................................35

1.3.5.2- Calcium in plant physiology.............................................................................................35

1.3.6) Magnesium..............................................................................................................................37

1.3.6.1- Mineral and availability...................................................................................................37

1.3.6.2- Magnesium in plant physiology.......................................................................................38

1.3.7) Sodium.....................................................................................................................................39

1.3.7.1- Mineral and availability...................................................................................................39

1.3.7.2- Sodium in plant physiology..............................................................................................40

1.3.8) Lithium ‘as the target metal’...................................................................................................42

1.3.8.1- Introduction.....................................................................................................................42

1.3.8.2- Lithium metallurgy...........................................................................................................42

1.3.8.3- Properties........................................................................................................................44

1.3.8.4- Occurrence......................................................................................................................44

1.3.8.5- Toxicity of lithium............................................................................................................46

1.3.9) Discussion................................................................................................................................48

Chapter Two: Experimental......................................................................................................................49

Methodological approach....................................................................................................................49

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Phase one: Formulation of growth media...........................................................................................53

2.1.1) LMT homogenous mixing.….....................................................................................................53

2.1.2) LMT drying process…...............................................................................................................53

2.1.3) LMT moisture content………………………………............................................................................53

2.1.4) LMT organic matter determination……….................................................................................54

2.1.5) LMT classification…..................................................................................................................54

2.1.6) LMT texture determination……................................................................................................55

2.1.7) LMT pH determination….........................................................................................................57

2.1.8) LMT salinity determination…...................................................................................................58

2.1.9) Peat moisture content determination………............................................................................59

2.1.10) Homogenized peat pH determination…………........................................................................60

2.1.11) Municipal biosolids pH determination…….............................................................................60

2.1.12) GM pH determination………………………...................................................................................61

2.1.13) ORP determination potential (Eh) of different TPB sample mixes........................................62

2.1.13.1 Introduction.......................................................................................................................62

2.1.13.2 Procedure..........................................................................................................................62

2.1.14) Salinity (EC) levels of different growth media mixes............................................................62

2.1.14.1 Introduction.......................................................................................................................62

2.1.14.2 Procedure..........................................................................................................................63

2.1.15) Discussion..............................................................................................................................64

Phase two: Chemical analysis of growth media...................................................................................66

2.2.1) Introduction.............................................................................................................................66

2.2.2) Atomic absorption spectroscopy.............................................................................................66

2.2.3) Metal extraction procedure from LMT, peat and de-B sample..............................................68

2.2.3.1- HCL acid extraction procedure for lithium and other minerals......................................68

2.2.4) Lithium measurement.............................................................................................................68

2.2.5) Magnesium measurement......................................................................................................69

2.2.6) Iron measurement..................................................................................................................69

2.2.7) Calcium measurement............................................................................................................70

2.2.8) Sodium measurement.............................................................................................................70

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2.2.9) Chromium measurement........................................................................................................70

2.2.10) Potassium measurement.......................................................................................................71

2.2.11) Vanadium measurement.......................................................................................................71

2.2.12) Conclusion.............................................................................................................................76

Chapter Three: The Column procedure....................................................................................................79

3.3.1) Introduction.............................................................................................................................79

3.3.2) General assembly....................................................................................................................80

3.3.3) Peat homogenization process.................................................................................................80

3.3.4) Biosolids dewatering...............................................................................................................80

3.3.5) LiCl amended procedure.........................................................................................................81

3.3.6) Homogenous mixing................................................................................................................81

3.3.7) Experimental procedure..........................................................................................................81

3.3.8) Leachate digestion procedure in hydrochloric acid.................................................................83

3.3.9) Organic fertilizer analysis........................................................................................................83

3.3.10) Metal content analysis per group basis and results..............................................................84

3.3.11) Discussion..............................................................................................................................99

Phase Four: Impact of EK on GM metal content determination........................................................100

3.4.1) Introduction...........................................................................................................................100

3.4.2) Electrokinetics cell description..............................................................................................101

3.4.3) Experimental process and results..........................................................................................103

3.4.4) Discussion..............................................................................................................................113

Chapter Four: Phyto-sequestration/stabilization...................................................................................114

Phase One: Phytoremediation...........................................................................................................114

4.1.1) Introduction...........................................................................................................................114

4.1.2) Sowing plant seeds in different growth media.....................................................................114

4.1.3) Seeding on five different GM mixes......................................................................................115

4.1.4) Harvesting process................................................................................................................116

4.1.5) Drying process.......................................................................................................................119

4.1.6) Storing process......................................................................................................................119

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4.1.7) Plant leaf analysis for lithium................................................................................................120

4.1.8) Plant leaf dry ashing process.................................................................................................120

4.1.9) Analysis for remaining minerals in plant leaf tissue sample.................................................121

4.1.9.1 Introduction......................................................................................................................121

4.1.9.2 Procedure..........................................................................................................................121

4.1.10) Plant stem analysis procedure for extraction of lithium.....................................................121

4.1.11) Analysis for remaining minerals in plant stem tissue samples............................................122

4.1.12) Plant root analysis procedure for extraction of lithium......................................................122

4.1.13) Analysis for the remaining minerals in plant root samples.................................................123

4.1.14) Metal analysis in rhizosphere..............................................................................................123

4.1.15) Mass balance calculation.....................................................................................................130

4.1.16) Effectiveness of the different TPB media mixes..................................................................130

4.1.17) Parameters of efficiency......................................................................................................131

4.1.18) Discussion............................................................................................................................134

Phase Two: Monocotyledonous plant phyto-extraction/stabilization analysis................................139

4.2.1) Introduction..........................................................................................................................139

4.2.2) Experimental process and results.........................................................................................141

4.2.3) Discussion..............................................................................................................................157

Chapter Five: Conclusion and Future work recommendations...........................................................148

Appendix...........................................................................................................................................151

References.........................................................................................................................................153

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Chapter One: Statement of the problem

Excessive anthropogenic activities resulted in the degradation of the biosphere. Mining industrial

discharge, smelting of metalliferous ores, domestic sewage and agricultural runoffs aggravated the

situation producing toxic pollutants. Counterbalancing this situation enabled the enactment of strict

environmental policy guidelines and regulations. Fulfilling theses guidelines paved the way for the

development of novel green technologies.

Lithium as a cornerstone component of green technologies, its usage and production skyrocketed. It is

mined from brine or rock sources and rendered in chlorite or carbonate form. Its annual increase rate

reached 5.1% or around 25 tons in 2008. It is projected to reach 7.7 tons in year 2020. Moreover, its

predicted increase will come from its usage in electrical vehicles (EV-s). According to Mohr et al. 2012

an EV battery of capacity 20kWh encloses 3 kg of lithium.

As a result the environmental flow of lithium will dramatically increase, added upon secondary

sources like pharmaceuticals products, lubricants, certain chemical and dyes. As a result it will have its

negative impacts on human quality of life and ecosystem biodiversity alike. Moreover, in some lithium

dissipative applications its recovery is extremely difficult due to the absence of technical or economic

reasons. This might lead to bioaccumulation in nature and end up in human food chain.

According to Fellet et al. 2011 and Grangera et al. 2011, lithium extraction process produces

hazardous heavy metals in its tailings. As an example elevated amounts of vanadium, chromium, iron,

etc. In addition, under certain environmental conditions they might get weathered producing toxic

effluents. This will have detrimental impacts on ecosystem biodiversity.

Having stated the negative impacts of lithium mine tailings as a major source of toxic heavy metals.

Added upon its very low fertility, minimum water holding capacity and unfavorable physical structure,

urged a novel way of approach in rendering it suitable for reclamation and vegetative growth

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purposes. Through the addition of one or more additives or ingredients that will improve its stability,

fertility and water holding capacity.

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Objectives

The research was centered on the hypothesis that a hyperaccumulator plant has in vivo ability to

phytoextract and phytostabilize lithium, inside its physiological parts. It was supposedly grown

homogeneously, in a heterogeneous growth media. In lieu with heavy metals that might have been

present in its rhizosphere.

Detailed objectives

To investigate the hyperaccumulator plant (e.g. Brassica juncea) ability to phytoextract and

phytostabilize lithium in a wide pH spectrum.

To evaluate the effectiveness of B. juncea’s phytoextraction and phytostabilization of lithium, in lieu

with vanadium and chromium present in the growth media.

To analyze the vanadium, chromium and lithium phytoaccumulation in different plant physiological

parts of the Brassica juncea.

To analyze the different heterogeneous rhizosphere formulated in terms of macro/micronutrient

storage and availability to the hyperaccumulant plant uptake.

To verify the suitability and effectiveness of the different growth media for phytoextraction and

phytostabilization purposes.

To evaluate the monocotyledonous plant ability to phytoextract and phytostabilize lithium and

chromium in the most favorable growth media.

To compare between the monocotyledonous and the Brassica juncea hyperaccumulant plants, in

terms of botanical efficiency parameters in the most favorable growth media.

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Phase one: literature review

1.1.1 Introduction to phytoremediation

The importance of detoxification of the contaminated sites is taking the front seat of action

nowadays. The traditional environmental engineering measures are very costly and impacts negatively

on environmental biodiversity. It is roughly estimated that the cost of cleaning an acre of soil in the

United States costs over a million dollars. Regarding this astrological cost and biodiversity loss, favors

phytoremediation as a viable alternative. [1,2,3]

The process of extraction heavy metals or organic pollutants from soil, water and air media through

the usage of green plants defined as phytoremediation. In common terms it is referred to as

environmental restoration. However, biologists have classified certain plant families that have special

abilities that entitle them suitable for remediation. The science of phytoremediation is very recent and

evolving very fast. Moreover, the phytoremediation process is a ‘marriage’ between numerous

multidisciplinary approaches like molecular chemistry, biology, soil science, plant physiology, genetics,

plant selection, etc. [4,5]

According to Marchiol et al. [6] the ideal hyperaccumulant plant should have the following traits. First

it must be able to hyperaccumulate the desired metallic element more than one percent of its dry

weight basis. Second it must tolerate the desired heavy metals without showing clear toxicity

symptoms. Third it must be a fast grower, high biomass producer and easily harvestable. [6]

Hyperaccumulant plants are capable of phytoextraction of heavy metals like Cd, Cr, Pb, Co, As, Se, Hg

and U. They might have no biological benefits to the plant itself. These plants are also capable of

storing heavy metals inside their upper or lower harvestable physiological sites, leading to the removal

of the targeted heavy metals from biosphere. The harvested plants are dried and ashed. The recovered

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heavy metals are reused or recycled if the technology permits; otherwise its ashes are buried in a

landfill. [4]

The hyperaccumulant plant ability is limited to the rhizosphere level of the contaminated soil profile,

which is determined by the root penetration depth. However, its advantages are reflected in its ability

to preserve the environment and surrounding ecosystem in comparison with different in or ex-situ

engineering procedures. Phytoextraction typically costs 40% to 50 % less than any other engineering

interventions, like precipitation, flocculation, sedimentation, ion exchange, reverse osmosis and

microfiltration. Consequently altering and degrading the biodiversity at the expense of contaminant

removal [7]. On the contrary, phytoremediation is easier to manage, it is an autotrophic system that

produces large biomass that requires little attention, nutrient input and is generally approved by the

public perception primarily to its aesthetic and eco friendly approach [8]. Some common hyper-

accumulators or phytoextractors are Brassica juncea (Indian mustard), Pelargonium (geranium) and

Helianthus annuus (sunflower); and many others.

Phytoremediation includes phytoextraction which is the extraction of the pollutant or the heavy metal

from soil. Phytodegradation or phytotransformation refers to the symbiotic relationship between the

rhizospheric microorganism and the hyperaccumulant plant roots. That decomposes the organic

pollutant to water and carbon containing compounds that are later used as plants nutrient.

Rhizofiltration is referred to plant root uptake of pollutants from aquatic environments.

Phytostabilization is the usage of the extensive root network of the hyperaccumulant plant to stabilize

the soil profile and render the pollutants immobile. Thus decreasing the effects of erosion and leaching

possibilities of the pollutant in the environment. According to Salt et al. [9] they proved that

phytovolatilization is also possible. It is defined as the absorbance of pollutants or heavy metals from

soil or aquatic media and its direct volatilization towards the atmosphere.

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The transport cycle starts from the roots of the hyperaccumulant plant. The pollutant passes through

different channels and membrane gates towards the upper physiological parts like the leaves, flowers

and the seeds. According to Baker [10], a few of the plants posses what is referred to as plant-mineral

barrier, which enables it to distinguish between different metals for uptake or not. [10]

Sometimes chemical similarities play a pivotal role in mineral uptake and storage. Besides absorbing

different micro or macro nutrients for growth, maintenance and reproduction purposes, the hyper

accumulator plants posses the ability to accumulate more than one percent of its dry weight basis the

toxicant concerned. [10,11]

According to Chaney et al. [12], it is possible to accelerate the whole process of phytoextraction

through different plant breeding programs. Selection, cross breeding and genetic engineering

processes, which might pave the way for commercialization of the naturally occurring hyper

accumulator plants.

Hyperaccumulator plants mostly belong to Brassicaceae (ex. Indian mustard), Euphorbiaceae (ex.

Phyllanthus), Asteraceae (ex.pantaclia) and Fabaceae families. Presently at least forty five families are

classified as phytoextractant. [12,13,14,15]

Phytoextraction is accomplished by two methods. First chelate assisted phytoextraction is applied to

heavy metals of upmost toxicity like chromium, arsenic, uranium, plutonium etc. According to Huang et

al. [16, 17] and JØrgensen [18], they showed that there are not suitable plants to extract Pb, Cd and U

from soil in sufficient amounts. They are usually accumulated around 0.01% to 0.06 % of their dry

biomass. Moreover, synthetic chelates like EDTA (Ethylenediaminetetracetic acid) was applied to lead

its phytoaccumulation increased 100 %, likewise to EGTA to cadmium and citrate to uranium. The

procedure of chelate application is done when the plant has reached its optimal growth stage.

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However; a chelate is added to soil and left for optimal metal extraction to occur. Afterwards the

plants are harvested, dried, burned and its ashes stored. [16,17,18]

It is estimated that a specific hyperaccumulator plants can extract between 180 to 530 kg∙ha ¯¹ of lead

per year, making a 2500 mg∙kg ¯¹ contaminated site, decontaminated achieved in less than ten years of

continuous planting. [17,19]

Besides its numerous advantages the chelate assisted phytoextraction, has numerous disadvantages.

As an example it increases the mobility of the contaminant in the soil profile, which might lead to

increased leachate leading to ground water contamination. Therefore, a careful examination and

assessment of the proposed site is of upmost importance. The second approach is the continuous

phytoextraction method which was followed throughout the research. Basically it is based on the

specific hyperaccumulant plant to extract the desired amount of the toxicant throughout its life cycle

from early germination to full maturity and early senescence. The continuous phytoextraction process

appear to be cost effective, non intrusive, socially accepted, aesthetically pleasing phytotechnology

with great potential for remediation of heavy metal polluted soils [20]. Thus the accumulation of more

than one percent of its dry mass basis was regarded as a benchmark of success. [21]

Besides being based on its genetics and physiological traits, hyperaccumulator plants are slow

growing, with somehow low biomass producing and the lack of specificity for a specific heavy metal is a

major drawback. In order to overcome the previously mentioned shortfalls breeding, selection and

other genetic engineering procedures are applied. [22]

Up to now, the assessment of success of a designated phytoextraction process was placed on the

pollutant removal. However, the ultimate objective of a phytoextraction process must be not only to

remove the pollutant from the soil profile but also to restore soil vigor. Thus rendering the soil profile

capacity to re-function again as a vital living ecosystem, to sustain biological growth, promote the

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quality of air and water environments, and maintain plant, animal sustainability and overall husbandry.

[23]

Hence, indicators of soil health must be routinely assessed in terms of botanical efficiency

parameters. Its activity, size and biodiversity of microbial communities must also be taken under

careful consideration. This paves the way for a truthful assessment of success or failure of a phyto-

reclamation and extraction approach and its implications as a whole. [24,20]

1.1.2 Prerequisites of successful ecological restoration

Successful ecological restoration of mine tailings depends on many factors. It must first prevent its

erosion and further degradation. However, many mining sites are characterized by increased acidity.

Second it is nutrient deficient and has a poor textural structure is a major drawback, which impedes

proper vegetative growth [263].

A noval approach was tailored in the form of addition of additives that were mixed with the lithium

mine tailing mass. They were characterizes as being cheap, abundant and readily available. The added

additives were the municipal biosolids and peat that compensated the shortcomings of the LMT. The

latter improved its texture and water holding capacity and the prior improved its living biota.

1.1.3 Brassica juncea “ hyperaccumulant plant”

The Brassica juncea Czern variety was chosen for the study because of its high phytoremediation

potential [28,9]. It has a high efficiency for accumulating numerous heavy metals, in its different

physiological parts [9]. Furthermore, the Brassica species are adaptable to a wide range of

environmental conditions and to different cultivation processes. [11, 28, 19, 29]

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On the plant side, the hyperaccumulant plant has a high resistance to heavy metal toxicity. On the

rhizosphere level it is known to form different symbiotic relationships within the growth media biota

that facilitates metal uptake and proper storage inside its harvestable biomass. [294]

Genetically the Brassica juncea family is tetraploid, thus having the double number of chromosomes,

which makes it very suitable for genetic breeding and selection procedures. For example the B. juncea

(AABB genome, 2n = 36), B. rapa (AA genome, 2n = 20) and B. nigra (BB genome, 2n = 16).

Finally China is considered the original region and varietal homeland with its highest level of

differentiation around the province of Sichuan. [27]

1.1.3.1 General description

Brassica juncea belongs to the family Brassicaceae and genus Brassica or commonly recognized as

the mustard family. Despite the variance between the brassicas and the mustards, but throughout the

research thesis the umbrella term of Brassica as a general term for all species will be used. The family

name reflects the shape of its yellow flowers that have four diagonally opposed petals forming the

shape of a cross. Its lower leaves are deeply lobbed and upper leaves are narrow and entire in pale

green color, with hairy like outgrowth from it. The leaves and leaf blades ends with a petiole. It is

worth mentioning that Brassica juncea is an annual crop, cool season, high biomass producer with

edible leaves. In addition, the genus Brassica makes a major contribution to human diet and to

livestock feeds. Moreover its oil is appreciated throughout the world. [25]

Mature plant can reach up to three feet high and produce seed pods that enclose 2.5 cm to 5 cm in

length yellow or brown sickle shaped seeds in sac like outgrowths. [26]

The US cultivars are called Southern giant curled, Florida’s as Broadleaf with leaf length reaches up to

0.6m wide. Asian cultivars are called green in snow, like the Osaka purple leaf with distinctive red

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leaves with white veins, Tai Tau Choi which is very popular in china mostly grown for its roots, which

resembles the turnip. In India the very well known varieties are Laha, Lahi, Lahta and Desi Rai. The

Abyssinian mustard (B. carinata) is planted widely in Ethiopia’s highlands and Eritrea. European

cultivars are French brown, Bargonde, Tilney, etc.

1.1.3.2 Origin and cultivation

According to Woods et al. [28] Brassica family plants domesticated were first a few centuries ago in

the far eastern Himalayan region. Later on it was spread to Europe and the Americas. Its edible parts

are above and below ground like its leaves, flowers and seeds as well as its roots. In the western world

it is regarded as a spice crop while in some parts of Asia it is considered as an essential source for

cooking oil.

In Canada its cultivation fields are mostly located in the Prairie Provinces like Manitoba, Saskatchewan

and Alberta reaching up to 41000 ha of yield range from 900 to 1235 kg∙ha¯¹. The principal growing

countries are Bangladesh, Central Africa, China, India, Japan, Nepal and Pakistan, as well as some parts

of southern Russia. [28]

Brassica plants family cultivation is considered as one of the easiest and less time and effort

consuming. Planted usually in early spring or in autumn like done in Florida during the months of

September or October and as a secondary crop in January. Major pests and diseases that affect its yield

are aphids, cabbage worms, etc. It is considered as a ‘hardy’ plant, as it can withstand extreme

temperature fluctuations as low as – 4 ⁰C up to 29 ⁰C. Likewise to extreme rain precipitations of 500 to

4200 mm and to a wide pH spectrum of 4.3 to 8.3.

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Like any other oil and glucosinolate producing plant the Brassica family needs proper nitrogen to

sulfur of 7:1 N/S fertilizer application. It is usually in the form of potash applied 50 to 75 kg∙ha¯¹, which

is regarded as its comfort zone needs. [29]

Gastronomically, its leaves are consumed in raw salads or cooked like spinach. In Kashmiri and Bengali

cooking it is usually pickled and usually is referred to as ‘Hum Choy and Sajur Asia’. Its seeds are used

as a primarily cooking oil source in some parts of India, Nepal and Pakistan. Moreover, low

glucosinolate and erucic acid content varieties are used as additives for cattle, pig and chicken feeds.

[ 26, 28, 30]

Finally, the genus Brassica engulfs fascinating varieties of sometimes distinct and complex species

like, B. hirta the white flowered mustard, B. juncea, or the brown mustard, B. napus as the rutabaga,

Siberian kale, rape seed (canola oil plant), B. narinosa as the broad beaked mustard, B. nigra as the

black mustard grown for, B. oleracea, as the cauliflower, the broccoli, the Brussels sprouts, the

cabbage, the collards, the kale and the kohlrabi and finally the B. rapa the turnip, broccoli raab,

Chinese cabbage and the Chinese mustard. [26]

1.1.3.3 Harvesting

The Brassica juncea’s growing period stretches for sixty days, depending on the variety and weather

conditions. The plant usually harvested before full maturity and fruiting stage, in order to avoid fully

ripe and seed shattering. Its harvesting usually achieved early morning, when the entire plant is pulled

manually in regions where labor is cheap but otherwise it is achieved mechanically. Entire plants are

tied together and left to dry for 4 to 10 days. Extraction of the oil from its seeds is done through rotary

mill, expeller or hydraulic processes. Its green leaves are immediately refrigerated for freshness and

quality purposes, packed and used as an ingredient in fresh salads and other edible dishes. [31]

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1.1.3.4 Benefits

The Brassica plants are sometimes used as a cover crop for their rapid growth, very efficient nutrient

uptake and storage inside its biomass and fast canopy closure [32]. Lately some species of Brassicas

have gained renewed interest in their ability for pest management purposes as biofumigant. Its root

exudates biotoxins or metabolic byproducts that are believed to contain active ingredient of sulfur like

thiocyanates that are regarded as repellent and even toxic to soil borne pathogens and pests, like

nematodes, fungi based pests, insects and some types of weeds [33, 34, 35]. Thus it is usually

incorporated in soil during tillage and other agricultural activities, usually on the onset of the pest life

cycle. [31]

Brassicas are also used as winter or even as a rotational crop in vegetable or mixed planted with

orchard trees. Moreover, it increases soil fertility through the increase of soil nitrogen content.

As a fast grower and a big biomass producer, it prevents soil erosion, if used as a cover crop. It is

estimated that Brassicas under normal circumstances can produce up to 8000 lb of biomass per acre,

but under stress conditions the biomass production mass decreases considerably.

Brassicas are very well suited for nutrient capture specially nitrogen, because of its dense upper

physiology [29]. Its roots may reach six feet deep, which plays a pivotal role in soil aeration, aggregate

formation and stabilization. [36, 29, 31]

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1.1.3.5 Diseases

Brassica family plants are effected from Rhizoctonia (canker and black scurf), Verticillium (common

wilt), Spongospora subterranea (powdery scab) and Streptomyces scabiei (common scab) are its

common “enemies”, inflicting serious damage and even plant biomass loss. Nematodes affect greatly

its rhyzosphere like Meloidyne chitwoodi (Columbia root knot nematode) and Meloidogyne hapla

(northern root knot nematode) [37, 38].

As for weed infestation that might result in nutrient loss are mostly annual like the pigweed,

shepherd purse, green foxtail, kochia, long spine sandbur and barnyard grass. [39]

1.1.3.6 Folk medicine

It is believed to be recognized as a diuretic, aperitif, anodyne, emetic, rube facient and stimulant.

Indian mustard varieties are remedies for arthritis, foot ache and rheumatism [40]. Its seeds are used

for treatment of tumors in China, also in Africa it is used as galactogogue. Its sun dried leaves and

flowers are smoked in Tanganyika in order to pave the wave for spiritual connection. Its ingestion in

Africa and in other parts of the tropics imparts mosquitoes through body odor [41].

Its oil produce is used as a counter irritant and also as a stimulant. In Java the plant is used as an anti

syphilitic agent. Its leaves if applied on the forehead are believed to relieve headache [41].

In the Korean peninsula its seeds are used against cold, lumbago, rheumatism and stomach disorders

and as in regard to the Chinese traditional medicine its fresh leaves are consumed as a cure for bladder

inflammation or hemorrhage and its oil produce against skin eruptions and ulcers. [42]

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1.1.3.7 Chemical constituents

Brassica family plants are extremely rich in vitamins, minerals, oils and other important constituents

which are vital to human life; thus its leaves are high in Vitamin A and C as well as Iron. A 140 g of its

leaves provide to an adult human being with sixty percent of his or hers daily recommendation of

vitamin A and all the vitamin C requirements as well as the one fifth of the iron daily need; moreover, It

contains 24 calories, 91.8 g of water, 2.4 g of protein, 0.4 g of protein, 0.4 g of fat, 4.3 g of

carbohydrate, 1 g of fiber, 1.1 g of ash, 160 mg of Ca, 48 mg of phosphorus, 2.7 mg of iron, 24 mg of

sodium, 297 mg of potassium, 1825 μg of β carotene equivalent, 0.06 mg of thiamine, 0.14 mg

riboflavin, 0.8 mg niacin and 73 mg ascorbic acid. [44, 45, 46]

According to Pryde et al. [46] and Knowless et al. [44] a 100 g of its root sample is reported to contain

38 calories, 85.2 g of water, 1.9 g of protein, 0.3 g of fat, 8.8 g of total carbohydrate, 2.0 g of fiber, 3.8 g

of ash, 111 mg of calcium, 65 mg of P, 1.6 mg of Iron, 447 mg of K, 45 μg of β carotene equivalent, 0.05

mg thiamine, 0.12 mg of riboflavin, 0.7 mg niacin and 21 mg of ascorbic acid.

Moreover, according to Leung [43], a 100 g of its seed is tested to contain 6.2 g of water, 24.6 g of

protein, 35.5 g of fat, 28.4 g of total carbohydrate, 8.0 g of fiber and 5.3 g of ash. Seed sterols contain

19.2 % brassicasterol (9.1 % esterified), 23.6 % free campesterol (34% esterified), 57.2 % sitosterol

(55.2% esterified), 1.7% esterified Δ-5-avenasterol and a trace of Δ-7- stigmasterol. It also contains the

glucosinolate sinigrin (potassium myronate) and the enzyme myrosin (myrosinae), sinapic acid,

sinaprine (sinapic acid choline ester), fixed oils 25 to 37 % consisting mainly of glycerides of erucic,

eicosenoic, arachidic, nonadecanoic, behenic, oleic and palmitic acids, among others proteins like

globulins and mucilage.

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Brassica juncea also contains volatile components such as methyl, isopropyl, sec-butyl, butyl, 3-

butenyl, 4- pentyl, phenyl, 3-methyl thiopropyl, benzyl and β-phenylethyl isothiocyanates. [44, 45, 36,

46]

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Phase Two: Mechanisms of mineral uptake and storage in

hyperaccumulator plant

1.2.1 Phyto-uptake and accumulation mechanism

Certain hyperaccumulant plant varieties have naturally developed tendencies to hyperaccumulate

certain metals. According to Reeves [47], the elevated amount of metal accumulation is due to their

distinct traits of tolerance, genetic composition and adaptation that distance them from the ordinary

plant species. These kinds of plants are getting much attention lately and regarded as stepping stone

for upcoming value added plants. The hyperaccumulant plants have evolved unique physical traits and

distinct enzymatic secretions that enable them to detoxify and precipitate them in benign forms inside

their different physiological resting compartments. [47]

It is widely accepted that a few of the hyperaccumulant plants have developed special tissue ligands

that chelate with the heavy metal present in the rhizosphere area. According to Reeves [47] this trait is

so strong that it enables the leaves of Alyssum plant to concentrate high amounts of organic acids like

citrate, malate and malonate. Moreover, according to Andrew et al. [48], suggested that the

hyperaccumulant plant Alyssum possess an increased amount of histidine as a ligand that gets

complexed with Ni and subsequently translocated it throughout its physiology. [48]

According to Boyd et al. [49], the Straptanthus polygaloides (grey Brassica) has the tendency to

hyperaccumulate Ni in considerable amounts. Thus it can tolerate up to 500 μm of nickel without

showing any toxicity symptoms, inside its roots. Furthermore, it was verified that the high

concentration of Ni in plant roots behaved as a defense mechanism against pathogenic

microorganisms that might cause damage to its roots.

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According to Rauser [50], the chelating compounds are mostly composed of peptides with a general

structure of [………-GloCys] n-Gly; where n > 1. Thus the “marriage” between the chelating agent and

the metal enables the newly formed compound to leave the hyperaccumulant plant root system and

concentrate inside its different physiological compartments.

1.2.2 Toxic metal resistance mechanism

The continuous phytoextraction process that the research was anchored upon raised numerous

challenges to the hyperaccumulant plant. The plant has genetically developed certain traits and

abilities to concentrate the excess toxic metals inside its physiology without affecting it much. It is

assumed to be detoxified first and then stored. According to Rugh et al. [51], the mercury resistant

Arabidopsis thaliana hyperaccumulant plant oozes certain types of reductases that efficiently detoxify

mercury in a hydroponically grown system.

On the biochemical level different enzymes that interact each other with numerous triggering and

blocking mechanisms in the form of reductases, such as superoxide dismutase (SOD) and antioxidant

catalase, which detoxify heavy metals. According to Tomsett and Thurman [52], Jackson et al. [53],

Ernst et al. [54], Gwozdz et al. [55], proved that heavy metal concentration increased the SOD activity

to a certain plateau and decreased thereafter, leading to a kind of toxification symptoms to appear on

the plant tissues.

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1.2.2.1 Chelating process

Chelating agents like metallothionein (MT) and phytochelatin play a pivotal role in reducing the

concentration of free toxic metals in soil solution through the formation of aggregates. The most

common type of chelating agent is the MT-s, which is mostly gene coded with lower molecular weight

and cysteine rich polypeptides [56]. According to Murphy and Taiz [57], the MT production levels in A.

thaliana hyperaccumulator plant increased considerably due to the extensive cupper presence. As for

the phytochelatin, they possess a low molecular weight. It is enzymatically synthesized with rich in

cysteine peptides. It is well documented to bind to Cd and Cu [58, 59, 60]. These peptides are

responsible for cadmium detoxification and proper precipitation in different physiological parts of the

A. thaliana hyperaccumulant plant. [61]

1.2.2.2 Biotransformation and compartmentalization processes

It is the incorporation of toxic heavy metals into special storage sites in plant cellular level. However,

rendering them non toxic and non harmful to the hyperaccumulant plant physiology. A vivid example

is the detoxification and biotransformation process of selenium. According to Läuchli [62]; Astragalus

plant is able to withstand high selenium concentration due to its ability, to get the selenium

metabolized to seleno-cyteine and seleno-methionine products. Then gradually replacing cysteine

and methionine in protein biosynthesis cycle, as a result the funneling of selenium out of the

methionine biosynthesis pathway into non protein amino acids like methyl-seleno-cysteine and

seleno-cystathionine, which is regarded as the main reason for selenium detoxicity.

Arsenic is another toxic metal to plants which is the main ingredient of several organo-arsenical based

herbicides. According to Francesconi [63], marine macro algae incorporates arsenic inside its leaf

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vacuoles through differentiating it into different dimethyl-arsinyl-ribosides and certain types of lipids

depriving its presence in ecosystem thus rendering arsenic benign.

Cr is detoxified in some hypertolerant plants through reducing the toxic Cr (VI) to a lesser toxic form

of Cr (III). According to Storage [65] demonstrated that the uptake of Cr (VI) was an active one in

contrast to Cr (III) uptake. Moreover, it is verified that the hyperaccumulant plant spends energy in the

form of ATP in order to uptake Cr (VI) rather than Cr (III). This process paves the way for the

hyperaccumulant like Leptospernum scoperium to withstand high chromium concentration in soil

through passive absorption of Cr (III) rather than Cr (VI).

1.2.2.3 Cellular repair mechanism

It is believed that the plasma membrane repairs are done as the direct result of absorption of toxic

heavy metals. The repairs are performed by MT-s [64, 65]. According to Murphy and Taiz [57] in A.

thaliana plant, the Acyl carrier protein (ACP) and Acyl CoA binding protein (ACBP) are involved in lipid

metabolism, which are primary constituent of the plasma membrane. It is primarily responsible for the

repair of the damaged membrane. In addition, they are very specific to the presence of elevated

amounts of Cu in rhizosphere area. However high concentrations of metal tolerance are not enough to

entitle the designated plant varieties as phytoextractant but its ability to successfully uptake,

translocate and store them efficiently.

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1.2.2.4 Root and shoot uptake and accumulation

In the continuous phytoextraction approach of phytoremediation, the chelate assisted uptake of

metals play an essential role in the whole process. Most of the heavy metals are strongly bound to fine

particles of the soil, rendering unavailable to plant uptake. However, the increased chelate production

such as mugenic and avenic acids by the plant root tips increased their soil bioavailability [66]. For

example iron triggers their secretion like root ferric reductases reducing chelated Fe (III) to Fe (II) which

is readily absorbed by the root [67] [68] [69].

According to Crowley et al. [71] and Welch et al. [70], certain plants have the ability to acidify their

rhizosphere region by pumping excessive protons from their roots, to absorb heavily bound metals like

Cu and Mn. Decreasing soil pH renders strongly bound heavy metals; that are otherwise unavailable, to

become available for phytoextraction. On the plasma membrane level some transporters like Cu-COPTI

and Fe-(IRT4) chelate compounds are responsible for cupper and iron translocation like in Arabidopsis

thaliana plant forming what is referred to as phytosederophore complexes. [71, 72]

Once the metal phytosederophore complex enters the root system, it gets transported to the shoots

and further reaching to its leaves through its xylem vessels. According to Salt et al. [73], cupper is

transported via the xylem sap of the Brassica juncea plant by displaying biphasic saturation kinetics,

suggesting it was accomplished by specialized and metal specific transport processes. It is known that

xylem walls have a high cation exchange capacity [73]. This may impede the free metal movement

through it. Therefore the non ionic chelate complexes assist towards their transport and final

translocation, mostly in the form of Cu-citrate complexes [74].

According to Salt et al. [73], in the xylem system of Brassica juncea plant, the Cd is translocated

through its complexation with a chelating agent like organic acids. Moreover, according to Stephan et

al. [75] clearly proved that the presence of non protein based amino acids in certain hyperaccumulant

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plants that have the ability to form chelating like complexes with divalent metal ions like Cu, Ni, Co, Zn,

Fe and Mn. [75]

1.2.2.5 Phytovolatilization

Phytovolatilization is a unique ability that the phytoextractant plants possess, as a means of

detoxification of the heavy metal absorbed through volatilizing it to the atmosphere. According to

Lewis et al. [76], proved the postulate that selenium hyperaccumulant plant species were able to

phyto-volatilize it as a dimethyl diselenate. According to Lewis et al. [76,77] proved that even a non

hyperaccumulator plant like the alfalfa was able to volatilize selenium in a similar way.

Zayed and Terry [78] demonstrated that when antibiotic penicillin was added to hydroponically grown

Brassica juncea (Indian mustard), it inhibited selenium volatilization by ninety percent. They suggested

that certain types of root-bacteria symbiosis activities in the rhizosphere area assisted in reducing

selenium into volatile forms.

However, this said it is of immense importance to take under careful consideration the different

environmental regulations, restrictions and impacts of such a process on human life and ecosystem

biodiversity.

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Phase Three: Overview of metals in lithium mine tailing

1.3.0 Geological formation

The parent rock pegmatite belongs to the superior geological province in the Canadian Shield. It is

composed of meta-volcanic and consequently derived from meta-sedimentary rocks and synvolcanic to

late tectonic intrusive rocks. The original pegmatite is one of the numerous sub-horizontal pegmatite

sheets, which belongs to the well famous Bernie lake pegmatite group. It is hosted by a synvolcanic

metagrabbro intrusive. It is composed of eight discrete mineralogical zones with different ores of great

economic importance, such as tantalum, spodumene LiAl(SiO₆), cesium and rubidium, each occurring in

different zones. Since its discovery different geologists have proven that the pegmatite is the host of

more than eighty different minerals, some of which being rare earth minerals, such as Rb, Ta, Sn, Ti,

Nb, Li, F, Cs, etc.

For simplicity reasons the sequence of metal analysis in the lithium mine tailing were divided into two

groups. First the heavy metals containing metals like Cr, V and to a certain extent Fe. Second the

remaining metals that are found in it and regarded as beneficial for hyperaccumulant growth and

development like Ca, Mg and Na.

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1.3.1 Chromium

1.3.1.1 Introduction

Chromium is present in environment abundantly. However, in soil it ranges between 10 to 50 mg∙kg¯¹.

Depending on the parent bedrock material, like in serpentine soils its concentration can peek up to 125

g∙Kg ¯¹ [80].

Chromium belongs to group VI-B and its electronic configuration is Ar. 3d⁵ 4s¹. In nature the stable

forms of chromium is Cr (III) which is a primary constituent of ores like ferrochromite {FeCr₂O₄}.

Cr (VI) is found in ores like {K₂Cr₂O₇}, though its haxavalent form is more toxic. Even though, the

divalent chromium is relatively unstable and readily oxidizable to its trivalent form. The tetravalent

chromium form does not occur naturally, but represents an important intermediate state as a rate

determinant for the pentavalent form of chromium (CrO₄) ¯³. Its half-life duration is very small and

usually defies detection. [79]

1.3.1.2 Chromium as a contaminant

Chromium is a heavily exploited mineral. It is used in numerous industries like leather processing,

finishing, electroplating, as a cleaning agent and in the production of chromic acid. The haxavalent

form of chromium is usually used in numerous industries, like metal plating, wood preserving and in

tanning products. As a result to its wide spread use, the haxavalent chromium easily leaches into the

environment and threatens the well being of its surrounding ecosystem. As an example in India it is

estimated that annually 2000 to 3200 tons of elemental chromium ‘escapes’ into environment mostly

as a byproduct from different tanning industries. [82]

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In plants with high chromium toxicity is expressed as stunted growth, which renders the root inability

to elongate growth and differentiate, causing cell collapse and death due to the absence of water and

nutrients in sufficient amounts.

According to Vasquez et al. [83], Mishra et al. [84] and Jain et al. [85] the high toxicity of chromium

decrease cell turgor pressure leading to plasymolysis. In epidermal and cortical cells of the bush bean

plants. They also proved that in neutral pH levels of Cr (VI) which is a strong oxidizer, might cause

oxidative damage to cells leading to cell death. Moreover, the Cr (VI), which is less water soluble

compared to Cr (III). In hydrated forms it penetrates cell walls and gets precipitated as a metal

chelating agent form.

1.3.1.3 Phytoextraction of chromium

It is known that the toxic chromium forms have detrimental effects on plant growth and

development, especially in acidic rhizosphere conditions, which increases its mobility. According to

Huffman and Allaway [86] and Huffman et al. [87] proved that chromium is not regarded as an

essential element to plant growth. They proved that as low as 0.38∙10 ¯⁶ mM of chromium content did

not have any effect on plant growth. It gets absorbed through the plant root system through specific

carriers due to chromium’s structural similarity with essential plant minerals like K, Mg, P, Fe and Mn.

According to Cervantes et al. [88], Cr (VI) gets actively transported, through cellular carriers. On the

plant tissue surface level, it competes against essential anions such as sulfates. Furthermore, due to its

structural similarity, it competes for the binding sites of essential plant growth metals like Fe, S and P,

but opposite to its trivalent form, whose uptake is passive.

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According to Shanker et al. [89], chromium gets immobilized in plant root vacuole cells. Only 0.1 % of

the total chromium uptake gets translocated to shoots and seeds. On the contrary over 98% of the

absorbed chromium stays in plant roots.

According to Skeffington et al. [91], and Zayed et al. [90], used radioactive tracers to study the Cr ⁵¹.

They reported that chromium moves mainly in plant xylem system. According to Rout et al. [92],

different concentrations of 2 ppm, 10 ppm and 25 ppm of chromium in sandy soil samples observed

11%, 22% and 41 % reduction in plant height with respect to the control. Moreover, they interpreted

their findings as high concentrations of chromium successfully competes with essential plant nutrients

and sometimes takes their place on membrane carriers, resulted in less nutrients and water available

for plant growth. Affecting plant overall reduction in size and height, especially around its leaves where

reduction was as much as 50 %. Finally major symptoms of chromium toxicity in plants are burned like

leaf tips or alongside its margins.

Chromium uptake by Brassicaceae family plants like cauliflower, kale and cabbage are noted due to

their Sulfur loving properties. They are reflected in their ‘hunger’ for chromium, especially if it is

supplied as CrO₄ ¯² form, due to the structural similarity with sulfate. It is absorbed, concentrated and

stored mostly in plant roots, but only a minute amount gets anchored in its upper parts. [90]

Brassica species as a whole and the Indian mustard in particular have an unusual ‘appetite’ for heavy

metal like Pb, Cr, Cd, Zn and Cu storage in its roots and translocating lesser amounts towards its

vegetative parts. Finally phytoextraction of chromium is performed usually in contaminated soil but

virtually nonexistent on tailing amended samples.

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1.3.2 Vanadium

1.3.2.1 Introduction

Vanadium is ranked as the twenty third element in the Mendeleev’s periodic table preceding

niobium and tantalum. It has a silvery metallic appearance of symbol “V”, has an atomic weight of

50.9415 with a specific gravity of 5.96 and a melting point of 1929 ⁰C [94,95].

Vanadium as a metal is recognized as one of the hardest of the metallic elements. It is very soluble in

nitric and sulphuric acid and lesser in hydrochloric acid. Vanadium is rust resistant; in addition, to its

toughness, it is greatly exploited in steel production. As example a half percent increase in steel

content raises the tensile strength of steal from 7⅟₂ to 13 tons per square inch. [95]

Vanadium is also added to non ferrous alloys used as tools in different machinery. As a vivid example

the ball bearings and the crank shafts of the world famous Ford T-Model were partially made from

vanadium [95]. Aluminum added to vanadium provides additional strength in titanium alloys in missile

cases production, nuclear reactors and jet engine compartments [96].

Vanadium is the twenty second most abundant element found in the earth’s crust at a mean

concentration of 150 g∙ton¯¹, similar to zinc, copper and nickel [93]. It is found as a major constituent in

over fifty different mineral ores with variable concentration in petroleum products. [97]

Major world suppliers of vanadium are China, South Africa, Russia, United States and Canada. The

latter deposits are in the form of titaniferous magnetite form mostly concentrated in Pipestone,

Manitoba, Lac Dore’ and Bell River in Quebec provinces. [98]

Generally vanadium consists of two isotopes of 0.24 % V⁵⁰ and 99.76% of V⁵¹. The first isotope is

slightly radioactive with a half life of 3.9∙10¹⁷ years (94).

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In nature vanadium has extremely complex and ever changing chemistry. It can readily change and

evolve anionicly or cationicly under different physiological conditions. It has been verified that the VO⁺²

is more prevalent in acidic soil solution rather than the VO₃¯ and VO₄¯² species, which are prevalent in

more neutral or alkaline soils. [99]

Vanadium is a ubiquitous element present in higher plants in trace amounts and in animals and

humans in ultra trace amounts. In human physiology the vanadium concentrate ranges between 100 to

200 µg [100]. As early as the 19th century vanadium was recommended in dire human pathological

cases like pneumonia, malnutrition, anemia, diabetes and tuberculosis. [101, 102]

Finally the vanadate (+5) has a special ability to mimic the cellular insulin functions which paves the

way as being regarded as a viable alternative. In brief the insulin-mimicking actions of vanadate are

related to its ability to block several liver metabolic enzymes. In addition, to several muscle and

adipose tissue enzymes, which all united decrease cellular glucose level. Moreover, it forces several

anti insulin enzymes to cease action, thus decreasing bodily glucose levels. [100, 103, 104, 105].

1.3.2.2 Vanadium as a contaminant

Vanadium has an extremely complex chemistry and is regarded as extremely toxic. The above

mentioned complexity might be attributed to vanadium’s multiple oxidation, hydrolysis and

polymerization states. The oxidation states of biological interest are the (⁺³), (⁺⁴) and the (⁺⁵) states. The

first is stable in extremely acidic media {pH<2} and in complete absence of oxygen, yet in vivo it is rare

to occur. The second (⁺⁴) state is stable in acidic media too like its predecessor as blue vanadyl cation.

As for the (⁺⁵) state which has wide range presence is mostly present in acidic pH as well as in

physiological pH. It tends to aggregate into polymer complexes. At lower pH, V⁺⁵ is a powerful oxidant

and pre dominates as an orange colored decavanadate {V₁₀O₂₈H₅}. [106]

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Vanadium pentoxide is one of the most toxic forms to human physiology. It can cause inflammation

of the bronchi and trachea, severe irritation to the eyes, skin, pulmonary edema, systemic poisoning

(MSDS, 2010). It elevates heart beat, causes skin rush, cough, labored breathing, loss of body weight,

reproduction and developmental toxicity, if untreated might eventually lead to death. [107]

At human cellular level it is documented that vanadium inhibits phosphate metabolizing enzymes,

such as phosphohydrolases, phosphotransferases, DNA-polymerases, thymidilate synthetase and

glyceraldehydes-3-phosphate dehydrogenase. [108, 109, 110]

According to Fay and De Vasconcelos [109], increased concentrations of vanadium effects nitrogen

metabolism in higher plants by inhibiting nitrate reductases. In sugar beets it inhibits phosphatases,

glutamic-pyruvic transaminases and invertases from secretion completely. Vanadate inhibits plasma

membrane hydrogen {H⁺} translocating ATPase, which impairs nutrient uptake on the plant membrane,

deprieving its ability to maintain its cell turgor pressure and stomata opening, leading to cell collapse

and eventual death. According to Beffagna et al. [116] and Kasai et al. [117] vanadate inhibits

phosphatases, phosphoenolpyruvate carboxylase, fructose 2, 6 bisphosphatase and many other on the

plant cellular level.

Vanadium is a common element in the lithosphere. Its average concentration is estimated around 150

µg∙g¯¹. It is a common component of alkaline and argillaceous rocks. Soils close to industrial areas or

near coal or fuel generating power stations are usually highly contaminated with vanadium [111].

Thus said the V⁺⁵ valency is considered extremely toxic, but little attention is shed on it. The V⁺⁵ {VO₄¯³}

and Cr⁺⁶ {as CrO₄¯²} have similar chemical properties [112], indeed they both can exist under similar

environmental conditions. Therefore, if plant vanadium {V⁺⁵} concentration exceeds more than 2 µg∙g¯¹

it may cause chlorosis and stunts its growth [113]. According to Rehder [114] and Panichev et al. [115]

it reduces the amount of phosphates in plant physiology, when V⁺⁵ rich grass is consumed by the

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grazing cattle it will transform into H₂VO₄¯² which replaces PO₄¯³ from cattle bones leading to

numerous forms of deformities, deficiencies and eventual herd loss.

1.3.2.3 Phytoextraction of vanadium

Vanadium is regarded by botanists and soil scientists as an essential but trace plant nutritional metal

but, its function is still somehow obscure. According to Cantley et al. [118], Macara [119] and Biggs and

Swinehart [120] Amarita musaria, mushroom specie is one of handful of plants that does accumulate

elevated amounts of vanadium in their physiology.

Vanadium hyperextractant plants are scarce and virtually nonexistent or unknown under field testing

conditions. Thus revealing and documenting vanadium hyper-accumulating plants are extremely

important, especially inside mine tailing containing growth media circumstances.

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1.3.2 Iron

1.3.3.1 Mineral and availability

Iron weights around 5% of earth’s crust and it is present in all soil types without exception. Fe is

present in minerals like olivine, biotite, etc. located in the centre of the octahedral in di or trivalent

form [164]. The Fe⁺³ easily oxides and has a low solubility due to its fast precipitation as Fe⁺³ +3OH

↔Fe (OH)₃ which is a solid [165].

Iron influences soil color from yellowish brown to fully brown in tropical well drained soils as hematite

form {α-Fe₂O₃} [166]. On the other hand, Ferrihydrate {HFe₅O₈.4H₂0} is regarded as a major source for

plant iron uptake. According to Chen and Barak [167], the solubility of iron oxides/hydroxides

decreases in the following order; Fe(OH)₃ amorphous > Fe(OH)₃ in soils > γ-Fe₂O₃ maghaemite > γ-

FeOOH lepidocrocide > α-Fe₂O₃ haematite > α-FeOOH goethite.

In alkaline soil Fe⁺³ solubility decreases as much as a thousand fold time for each unit of pH rise.

Forming Fe (OH)₂⁺, Fe(OH)₃ and Fe(OH)₄. On the contrary in acidic soils iron solubility and availability to

plant needs increases. [168]

In anaerobic soil conditions, Fe⁺³ is reduced to Fe⁺². Furthermore, the whole process is governed by the

presence of anaerobic bacteria, which uses Fe oxides as electron acceptors in their respiration

pathways [169], thus reducing the ferric {⁺³} to ferrous {⁺²} as follows;

Fe (OH)₃ + e¯ +3H⁺ ↔ Fe⁺² +3H₂O [170]

One obvious outcome from the above mentioned equation is that the reduction of Fe⁺³ to Fe⁺² is

associated with the consumption of H⁺, leading to an oxidation process of Fe⁺² to Fe⁺³.

The most important characteristic of iron in botany science is its ability to form organic complexes

called siderophores. They are formed as an interaction between the bacteria as well as fungi and

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plants; which are sometimes referred to as phyto-siderophores, which aid in numerous element

transport and availability. [171,172]

According to Masalha et al. [172] and Becker et al. [173] more than hundred distinct siderophores are

known, which are stable at various pH ranges. As an example a very well known siderophores is the

hydroxamic acid {R-CO-NH-OH} that binds with Fe⁺³ forming an ionic bond which is referred to as ferric

mono hydroxamate. [173]

1.3.3.2 Iron in plant physiology

The transport of Fe⁺² is generally accomplished into the roots by simple diffusion or by mass flow

phenomenon. It is translocated through the plasmalemma membrane, bound to Fe⁺³ reductases.

Moreover, the reductase accepts e¯ from the siderophores and disintegrates releasing Fe ⁺². Later on it

passes through special channels that lead it into the cytosol. The whole process takes place alongside

with the reduction of NADPH to NADP⁺ and H⁺ ions [174].

According to Takaji [175] and Takaji et al. [176], rice and barley roots excrete phyto-siderophores like

mugineic and avenic acid which are capable of mobilizing Fe⁺³, rendering it available to plant uptake.

Iron containing enzymes have an immense importance in rDNA synthesis. Furthermore, without

which the growth is depressed and senescence follows soon. Despite its immense abundance and

importance to plant growth and development iron toxicity and deficiency is characterized by failure of

chlorophyll production, chlorosis of younger leaves and further reduction of growth [177]. Maintaining

a good humus levels in soil is a way of optimizing the availability of iron. Finally iron toxicity is a serious

problem in rice plantations which might range between 300 to 1000 µg∙gr¯¹ of iron per dry weight. As a

result its leaves will be covered by tiny brown spots and chlorosis, the whole phenomenon is referred

to as bronzing. [178]

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1.3.4 Potassium

1.3.4.1 Mineral and availability

It is estimated that the mean potassium availability in earth’s crust is around 23 g∙kg¯¹. It is largely

bound to primary minerals and secondary clay particles. Its availability and absence depends on the

type of parent material. Weathering, time, topography and other soil formation parameters, play a

vital role in its presence or absence.

According to Laves [121], soil K⁺ content is closely related to illites, aluminum bearing chlorites and to

a lesser amount to smectites. The potassium element is a basic constituent inside the feldspars forming

its tetrahedral form. Thus the K⁺ is sandwiched between the Si-Al-O lines of the crystal lattice and held

tightly by covalent bonds [122, 123]. Weathering and presence of weak organic acids make potassium

available in lithosphere solution. It is also present abundantly in different types of micas which differ

from feldspars due to its Si-Al-O tetrahedral sheets, which contains a M-O,OH octahedral sheet in

which potassium occupy the hexagonal spaces.

According to Farmer and Wilson [124], the weathering process converts micas to secondary two to

one clay minerals by the following order; Micas (10% K) to hydro micas (6-8% K) to illite (4-6% K) to

transitional minerals (3%K) and finally vermiculites or montmorinolite (<2% K).

In mobility and fixation terms clay minerals fix potassium in dry and moist conditions like in micas and

vermiculites. Other smectites fix potassium under dry conditions only [125]. The increase of the planar

surface (p-position) and the interlayer (i-position) increases potassium fixation ability on a clay surface.

According to Schuffelen [126] and Sparks [127], the three different K⁺ binding positions on illite have

the following Gapon coefficients; for p-position 2.21 (mol∙m¯³) ¯½, for the e-position 102 (mol∙m¯³) ¯½

and for i-position as infinite per (mol.m¯³) ¯½.

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Potassium is a major constituent in numerous soil structural elements, which is regarded as

unavailable for plant use. Secondly it is bound or adsorbed on the exchange sites of the soil colloids,

which might be available to plant uptake and use under appropriate circumstances. Thirdly dissolved

potassium in soil solution is regarded as the readily available source for plant use. According to Martin

and Sparks [128], in sandy loamy soils the exchangeable K⁺ ions are estimated around 1.72 mol∙kg¯¹

and the non exchangeable portion around 2.20 mol∙ kg ¯¹.

1.3.4.2 Potassium in plant physiology

Potassium is an essential macro nutrient in plant growth, flowering and fruit bearing stage. It plays a

pivotal role in different enzymatic secretions. In addition, potassium is an essential ingredient in starch

synthesis and the development of chlorophyll. Unlike phosphorus and nitrogen, which are regarded as

structural metals, potassium plays a catalytic role in different chemical processes. Potassium is

taken up by the root plasma membrane and transported through different membrane based

transporters.

According to Matthuis and Sanders [129], Fox and Guerinot [130] and Schachtman and Shroeder [131]

on the plasma membrane, there are two main types of transporters related to potassium uptake and

translocation. The high and low affinity transporters, the former being very selective for K⁺ and is

characterized by low Km which ranges between a few mM per m¯³. The latter being less selective with

a Km of 1 mM per m¯³, thus the potassium ion passes from the plasmalemma into cytosol and further

inside the cell. Moreover, it gets transported through the xylem and phloem transport cells. The whole

process is governed by the difference in electrochemical gradient and saturation kinetics.

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The benefit of potassium in plant growth is defined by its broad spectrum functions. It initiates

meristematic growth, water uptake, photosynthetates translocation, enzymatic activation in starch

synthase inside sweet corn [133] and ribulose biophosphate carboxylase in polypeptide synthesis in

the ribosome, paving the way for protein synthesis in different plant tissues. [134]

On the other hand potassium deficiency in plants does not produce immediate or instant symptoms,

but gradually reduces its growth and necrosis of older leaves. It decreases the turgor pressure, leading

to poor resistance to drought, salinity, and frost. It increases susceptibility to fungal, microbial and viral

diseases leading to premature cell death. [135]

On the cellular level its deficiency leads to the collapse of chloroplast [135], mitochondria [136] and

retards cuticle development [137], leading to an overall low yield. According to Glynne [138] and Goss

[139] its deficiency symptoms can be easily corrected through proper fertilizer combinations that

might prevent lodging of maize and wheat. It is observed that a relative increase in K⁺ will increase

plant resistant to Fusarium wilt in banana, brown wilt (Puccinia hordei) in barley and brown spot

(Ophiobolus miyabeanus) in rice.

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1.3.5 Calcium

1.3.5.1 Mineral and availability

Calcium (Ca) concentration in earth’s lithosphere is estimates around 36.4 g.kg¯¹. It is one of the most

abundant minerals ever found. Calcium occurs as Ca bearing Al-silicates like feldspars and amphiboles.

As well as in calcium phosphates and calcium carbonates [140] moreover, in calcite {CaCO₃} or

dolomite Ca Mg (CO₃)₂. The weathering process is the major supplier of calcium into soil solution thus

its solubility and availability is as follows;

CaCO₃ + CO₂ + H₂O → Ca (HCO₃) → Ca⁺² + 2HCO₃¯

Calcium is recognized for its unmatched abilities in soil buffering and rendering it suitable for

agricultural crops growth and development [140]. As an example plant roots excrete increased

amounts of H⁺ ions that acidify the rhizosphere and renders heavily bound trace elements soluble and

available for plant uptake. The presence of calcium based soils have the ability to quickly reverse the

direct effects of acidification and render the pH at the optimum growth level.

1.3.5.2 Calcium in plant physiology

In dried plant calcium content ranges between 5 to 30 mg∙g¯¹. Generally calcium concentration is ten

times higher that the potassium. It is absorbed by the endodermis of the young developing root tip.

[141]

Despite its considerable presence in plants, calcium concentration differs in monocots in lesser

amounts rather than in dicots in greater amounts. According to Lonergan and Snowball [142], proved

that the rye grass needs are estimated around 2.5 mM∙m¯¹, where 100 mM∙m¯¹ to tomato (a dicot) of

available calcium.

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The reason behind such a difference was presumed to be due to the higher cation exchange ability in

the tissue cells and elevated amounts of carboxylic radicals in their cell walls. [143]

The Ca⁺² pathway in plant entry and translocation starts from the non suberized apoplast of the root

cell tips. Then through the process of facilitated diffusion it enters plasmalemma and furthermore,

through electro chemical gradient difference between the apoplast and the cytosol gets translocated

throughout the plant physiology. [143]

In plants, calcium is present as a free ion Ca⁺² or bound to radicals like carboxylic, phosphorylic,

hydroxylic groups. Its role is mainly to counter the cationic or anionic inorganic and organic

compounds. In seeds calcium is present in the form of salt inositol hexakisphosphate (usually referred

to as phytate). Calcium is often applied to the soil to release other nutrients by altering the soil acidity

(pH). Calcium is essential for the proliferation of soil bacteria. Calcium displaces sodium attached to

clay particles and holds clay particles further apart leading to its friability.

On cellular level calcium ions plays a regulating and stabilizing role for different membrane bound

ionic pumps like the Ca⁺²-ATPase and Ca⁺²/nH⁺ antiporters. They drive ions back and forth in the plant

cell vacuoles [144,145].

Calcium plays a pivotal role in different plant growth and development processes, through preserving

the integrity of the cell wall. Its deficiency on plants is reflected in reduced growth, browning of root

tips and early senescence [146]. It is also required in cell division, elongation, extension and

differentiation [147,148]. In addition calcium is responsible for fruiting and ripening processes through

the increased secretion of ethylene, which is secreted from cell wall membrane complex, outside the

cytoplasm [149].

Poovaiah and Leopold [150], Bush [143] and Lerchl et al. [151] showed that the Ca⁺² absence from the

maize leaves render it susceptible to early senescence and abscission of its leaves.

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On the cellular processes level calcium is regarded as an ionic balance, gene expression, mitosis and

secretion of numerous enzymatic secretions as well as an ion safeguarding the cellular homeostasis

situation.

Similarly, calcium deficiency reduces growth of the meristematic tissue, resulting in chlorosis and

necrosis of leaf margins. In Brussels sprouts (Brassica oleracea) calcium deficiency is categorized as

internal browning leading to internal rot [152]. Its deficiency causes the ‘black heart’ nutritional

symptom on the celery core [153].

1.3.6 Magnesium

1.3.6.1 Mineral and availability

Magnesium concentration in the earth crust ranges between 0.5 to 5 g∙kg¯¹. Magnesium is as an easily

weatherable element usually from biotite, serpentine and olivine minerals. In which its concentration

might reach up to 130, 250 or 240 g∙kg¯¹ of soil [154].

The plant beneficiary magnesium, constitute 5% of total magnesium present in soil solution. It

constitutes normally 4 to 20 % magnesium on total CEC available sites, presumably the average Ca⁺²

ranges on CEC sites is between 60 to 80 % [155,156].

Mg⁺² is not highly bound to clay minerals like K⁺ which is found between the 1:1 silica layers and

therefore resistant to leaching and plant availability. On the contrary magnesium is prone to leaching

and rapid loss from rhizosphere, which might reach up 30 kg∙ha¯¹∙year¯² [157].

Finally soil texture and parent material formation play an important role in magnesium storage or

loss. Sandy textured soils are prone to excessive leaching and loss. While the clayey based soils like

basaltic, petidolitic and dolomitic supply ample amounts of magnesium for plant uptake and usage.

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According to Grimme et al. [159] and Schimansky [160] magnesium uptake by plant roots occur due

to concentration difference gradient between the roots and the rhizosphere. Its entry and passage is

accomplished through the tonoplast and mediated by facilitated diffusion.

1.3.6.2 Magnesium in plant physiology

A well known location of magnesium in plants is in the centre of chlorophyll molecule. One of the

major tasks of Mg⁺² in plant physiology is the formation of bridge between the ATP and numerous

enzymes. As a result phosphorylation is catalyzed and transfer of energy occurs [161]. Likewise in

dephosphorylation v-pyrophosphatase based processes as well as in chlorophyll synthesis [145].

According to Travers [162], one of the fundamental functions of Mg⁺² in the nucleic acids is

safeguarding its structural and conformational integrity. In addition it is a major constituent in DNA and

rRNA which requires metallo-enzymes for synthesis alongside the divalent cation Mg⁺².

In particular most ribozymes require high concentration of Mg⁺², which is a prerequisite for efficient

binding of tRNA strand to the ribosome, but its absence deprives the polypeptide chain from synthesis

[163]. In addition to its numerous benefits, magnesium deficiency reveals chlorosis on older leaves and

spreads to younger ones. Magnesium deficient leaves fall prematurely, in celery its deficient leaves

show small dark spots on pale colored background [144]. Finally a 2 mg.g¯¹ of magnesium is regarded

as a threshold level for proper growth, development and fruit bearing stage. [106]

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1.3.7 Sodium

1.3.7.1 Mineral and availability

Sodium (Na) is derived from the Latin word sodanum, meaning a headache remedy, has an atomic

mass of 22.99 g∙mol¯¹ and a valence of (⁺¹). It is the sixth most abundant element comprising about 2.6

% of its crust [179]. It is found in different salt formation, which is usually easily dissolvable in water.

Therefore, its hydrated radius is 0.38 nm and its crystal ionic radius is 0.097 nm [180]. Sodium is not

considered as an essential plant nutrient except for those using the C4 pathway and halophyte plants.

[181]

Sodium has a similarity with potassium chemically and structurally. The potassium hydrated ratio is

0.33nm, which is very close to sodium. Its primary source is from irrigated water especially in arid or

semi arid areas. These types of textured soils contain considerable amounts of soluble salts like sodium

chloride, which hinders plant growth and development. [182]

Even though it is regarded as a non essential mineral to plant physiology, but it is extremely essential

to human as well as animal growth and development. It is regarded as the principal electrolyte in their

physiology and plays a pivotal role in maintaining the ionic balance of the body fluids and safeguards

against excessive loss of water from cell content [183]. The excessive sodium levels are mostly

associated with high salinity effects which deprive large areas of land for agricultural practices, leading

to fertility loss and desertion. [184]

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1.3.7.2 Sodium in plant physiology

According to Arnon and Stout’s [186] and Epstein [185] a mineral is considered essential when the

plant can not complete its life cycle, starting from its germination and ending by its seed production

and senescence. Second the desired mineral must have a specific and targeted action on the organism.

Third if it is a constituent of an essential compound, it is regarded as an essential element.

Based on the above mentioned definition, sodium is regarded essential for C4 plants like Atriplex

vesicarra, Kochia childsii etc. Without which chlorosis and failure to form flowers follow. Sodium is

needed in micro amounts (100 µm) to alleviate the above mentioned symptoms.

Sodium is generally recognized to promote growth on asparagus, barley, sprout, carrot, radish, rape

and Swiss chard [187,188]. However it is not detected directly in their metabolic processes, thus

remaining ‘discrete or hidden’. Sodium is suggested to get absorbed on plant root sites by the process

referred to as selective ion transport. It depends on metabolic energy derived from ATP breakdown

[189]. The overall absorbance of sodium depends on the presence of potassium in solution, leading to

a difference in gradient ratio. [190]

The absorbed sodium is translocated freely and stored in plant shoots in different concentrations. The

whole process of storage and translocation is referred to as natrophilic [191]. According to Greenway

and Osmond [192], the natrophilic plants avoid sodium toxicity due to their ability to efficiently

compartmentalize the excess sodium into their cellular vacuoles and use it as an inorganic osmoticum

for overall cellular ionic regulation. Even though, it is assumed that the reasoning behind sodium

storage in cellular vacuoles is due to the inability of the cytoplasmic ‘entourage’ to tolerate elevated

levels of more than 20 mM. Its excess might interference with the proper functioning of the

homeostatic process. [193,194]

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On the cellular level, sodium is proven to be beneficial to C4 plants. It plays a critical role in

regenerating the phosphoenolpyruvate (PEP) in the mesophyll chloroplasts of Amaranthus tricolor

plant [195]. Sodium takes part in chlorophyll synthesis [196]. According to Marschner [196], sodium

deficiency in C4 plants result in excessive accumulation of pyruvate in their mesophyll chloroplast and

inability to get it transformed to PEP.

On the enzymatic level sodium is generally regarded as a less effective mineral in activating various

enzymes, in comparison to other minerals like potassium and magnesium [197]. On the contrary at

elevated concentrations it triggers protein synthesis and oxidative phosphorylation to occur in vitro

experiments. [134,197]

Regardless of sodium concentration in rhizosphere that might be regarded as a potential toxicant to

plants roots. Most crops translocate it in minute concentrations and store it in their reproductive sites

like the seeds, fruits or even in other physiological compartments like inside its roots which might

sometimes be regarded as its edible part [198]. Like in wheat, rice, fruits, vegetables, tubers, carrots,

etc. The main reason behind it is its translocation system which is usually achieved through its xylem,

rather than phloem. [199, 200]

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1.3.8 Lithium: ‘as the target metal’

1.3.8.1 Introduction

Lithium is an ultra microelement, relatively little known and investigated. It is the third element in the

periodic table in group 1 A. Lithium does not occur freely in nature but it is bound in merely trace

amounts to igneous rocks like leidolite, pedalite, spodumene, amblygonite and many others. It is also

found in aquatic media.

Nowadays, lithium production and pricing sky rocketed once again. It is an integral part in the

production of rechargeable batteries in the form of lithium carbonate. Nowadays lithium based

batteries are found in human heart pacemakers buried deep inside the human chest, in television sets,

calculators, airframes and other structural components in the aerospace industry which consists of Al-

Cu- Li alloy of 2 to 3 % lithium by weight. [201, 202]

According to the United States Geological survey (USGS), in 2007 Chile was the leading producer of

lithium with 43% of worldwide production followed by Australia, 27%, China 12%, Argentina 11%,

Russia, U.S, Canada and Zimbabwe. Finally it is estimated that a pound of lithium carbonate is worth

around three Canadian dollars.

1.3.8.2 Lithium metallurgy

Lithium is strongly bound to rocky samples in minute amounts. The strategy of exploitation and

removal of lithium involves its conversion to carbonate, then to chloride followed by salt electrolysis.

[203]

In Canada lithium is specially mined around Lac du Bonet area in the province of Manitoba, in Quebec

there are numerous findings associated around Baie James, Eastman, Pontiac and around the Preissac

and Lac Corne areas. [204,205]

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1.3.8.3 Properties

The lightest of all metals, soft, silvery white in color. Its atomic number is 3 and its electronic

configuration is 1s² and 2s¹. A single atom of lithium consists of a nucleus in its innermost core,

surrounded by 2 electrons orbiting around it, which is inert. On the outer layer a single electron is

present which is readily given in order to obtain a state of stability, thus a (⁺¹) sign is designated.

Its geometrical arrangement represents a cubical lattice shape that renders it a perfect conductor of

electricity. Its atomic weight is 6.941 g∙mol ¯¹ and has one of the lowest densities a mere of 0.53 g∙cm³

at 20 ⁰C. [206]

Chemically, lithium is denoted as the first metallic element found in group IA; the alkali metals group,

followed by sodium, potassium, rubidium and cesium. Lithium is slightly harder than sodium but much

softer than lead. It readily reacts with water and even is able to absorb moisture from its surrounding

forming LiOH and hydrogen gas. Lithium’s melting point is 180 ⁰C and its boiling point is 1347 ⁰C. [207]

Lithium is stable in dry air, when the dew point is maintained below – 38 ⁰C. The lithium element

isotopes occurs as ⁶Li (7%) and ⁷Li (93%) forms. According to Sephton et al. [210], Suzuki et al. [209]

and Meneguzzi et al. [208], ⁷Li was produced during the cosmic big bang rather than the ⁶Li. Hence

according to them the ratio of ⁷Li to ⁶Li is an important indicator of galactic evolution in relation to

carbonates and phyllosilicates in rocky chondrites formations.

In a recent study that was conducted on different lithium isotopes revealed that a moderate

temperature of ~ 300°C enabled the retention of ⁶Li isotope in its solid phase. While on the contrary

the ⁷Li passed into the aqueous solution. However, lithium at room temperature reacts readily with

nitrogen forming Li₃N. [211, 210]

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1.3.8.4 Occurrence

Lithium in nature is in a combined form but in minute quantities. As an example it is associated inside

igneous rock formations like lepidolite (3.84 % wt), pedalite (2.09% wt), spodumene (3.73% wt),

amblygonite (3.44 % wt) and zinnwaldite (1.59 % wt). Lithium is also found in seawater, mineral springs

and sandy formations. It is estimated that lithium soil content is estimated around 13 million tons

while in sea its content is estimated at 230 billion tons.

Lithium is found in freshwater at different concentrations ranging from 0.1 ppb to 100 ppm, usually

higher concentration of lithium is accompanied with higher concentration of sodium. Hence it is

common to find lithium in fresh water content up to 10 ppb. In irrigated water its presence varies

between 2 to 5 ppm. At higher concentrations it may hinder plant growth and fruiting process.

Medically it is proven that elevated concentration of lithium in drinking water has a balneological

effect by decreasing the risks of coronary illnesses. Furthermore, a higher concentration of lithium in

drinking water has a positive influence on human nervous system, in disguising aggressive manners.

[212,213]

During human embryonic development, lithium levels reach its maximal values during the first

trimester of gestation period and subside afterwards. In animal studies lithium plays a significant role

in expansion of pluri-potential stem cell pool to new progenitor cells, leading to the formation of blood

cells. [214]

Moreover, an average daily intake of a 70 kg adult person in the United States ranges between 650 to

3100 μg of lithium. The green vegetables and drinking water are considered as its major sources. [214]

In human physiology, numerous trials have proven the presence of high concentration of lithium in

cerebellum, followed by cerebrum, kidneys, lungs, ribs, thyroid and liver.

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Astonishingly women physique surpasses men in lithium content by merely 10% to 20 %. [214]

Lithium is regarded as a non physiological cation. It has numerous in vivo effects. It became a

“household” medicine starting from the early 1950-s, when its carbonated form was believed to be

beneficial in treating different manic depression forms. The current hypothesis attributes lithium to its

ability to inhibit the enzyme inositol mono phosphate phosphomonoesterase. Under certain

circumstances it increases the stimulation of phospholipase C (PLC) coupled receptor systems in cells.

Eventually it may lead to the reduction of intracellular free inositol concentration, which may be

sufficient to diminish inositol phospholipid resynthesis and therefore the availability of the substrate

for PLC synthesis. [215, 216]

According to Del Rio et al. [217], assumed that the absence of PLC synthesis leads to inositol

phospholipids which generate a ubiquitous second messengers like inositol 1, 4, 5-triphosphate [Ins (1,

4, 5) P₃] and diacylglycerol, which effects human brain function. Nowadays, lithium bearing medicine is

widely prescribed. It plays a vital role against different dermatological and oncological diseases. [214,

215]

On inter or intra cellular level lithium biochemical effects appear to be extraordinary complex and

partly unknown. It is presumed that lithium is involved in numerous biological actions like inhibition of

adenacylate cyclase and the increases of GABA activity. It is also assumed to simulate parathyroid

activity, blocks vanadate bonding, and inhibits PGEI synthesis. On the other chemical level lithium is

linked with the increase in MAO (monoamine oxidase) enzyme synthesis which elevates mood [218].

However, it is medically proven that lithium enhances folate and vitamin B₁₂ transport and distribution

into L1210 cells. It is assumed that lithium might stimulate the production of new brain cells, which

might lead for a suitable cure against Alzheimer’s disease. [214]

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1.3.8.5 Toxicity of lithium

The effects of lithium toxicity on cultivated plants were first investigated by Kent in 1941. He was able

to prove its increased resistance led to disease resistance and stimulation of growth. A lithium toxicity

symptom varies in different plant genera. In fruiting trees above 0.05 ppm might be is considered toxic.

However, the main source of plant toxicity is the presence of lithium in irrigated water. Lithium toxicity

symptoms are difficult to recognize and differentiate from other type of necrotic spots, leaf curling or

chlorotic symptoms.

Generally speaking lithium injury reveals necrosis along the leaf margins, subsequent intervienal

chlorosis and leaf abscission [221]. It is well documented that lithium inhibits rhythmic movements of

pulvini and petals [222], disrupts normal pollen development by inducing symmetrical mitosis in the

microspores [223], blocking pollen germination to take place. [224]

According to Berridge [225], lithium affects the inositol depletion form cells which lead to the

inhibition of inositol monophosphatase synthesis. The stoppage of the inositol cycle leads to de-

signaling of cellular calcium and proper cellular membrane carrier activity. [223,224,226]

According to Boller [227] and Conegero et al. [228], higher concentrations of lithium stimulate

increased production and secretion of ethylene, salicylic and gentistic acids in tobacco plants. Its

excessive presence mimics the presence of a pathogen, triggering the start of the defense mechanism,

through induction of the 1-aminocyclopropane-1-carboxylate synthesis genes.

According to Smith and Blair [229], wheat powdery mildew (Erysiphe graminis) severity was reduced

as much as 11 % when wheat leaf area was fed with 8 g∙l¯¹ lithium chloride amended in fertilizer. In

addition Carter and Wain [230] analyzed the lithium sulphate increased the resistance of wheat root

seedlings against the E. graminis.

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In humans lithium overdose effects depend on its concentration especially when it is taken in

‘concert’ with alcohol or other illegal drugs. Moreover, lithium is used as a catalyst in ammonia/ alkali

synthesis method of the methamphetamines or commonly known as meth, from ephedrine. [231]

Its overdose symptoms are characterized by shakiness, vomiting and excessive thirst, frequent

urination, muscle weakness, seizures, blurred vision which might lead to an eventual coma. It is also

proven that the abuse of legal medications like Eskalith® and Lithobid®; which are used to treat bipolar

disorder or manic depression, might lead to lithium overdose.

However, lithium deficiency in humans might give rise to altered behaviors, like aggressiveness,

schizophrenic behaviors and homicide. After four weeks of clinical trials its supplementation resulted in

general happiness, friendliness and energy. [214]

Finally lithium’s ability to cause tetratogenicity (birth defects) in developing fetus is also a medically

proven condition. On a similar study of lithium carbonate on pregnant mice [232], numerous scientists

have reported that excess lithium might cause congenital defects and Ebstein’s anomaly; a rare cardiac

defect. [233]

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1.3.9 Discussion

According to the mining company data, the lithium mine tailing (LMT) sample was the produce, of the

direct result of the lithium extraction from different pegmatite sheets composed of meta-volcanic and

derived meta-sedimentary rocks and synvolcanic to late tectonic intrusive rocks. Internally the

pegmatite was composed of eight discrete mineralogical zones with different ores of economic

interest. The pegmatite rock was the host of more that eighty different minerals. The important ones

were stated as, spodumene {LiAl(SiO₆}, quartz {SiO₂}, feldspar {KAlSi₃O₈-NaAlSi₃O₈-CaAl₂Si₂O₈},

montebrasite {LiAlPO₄(F₉OH}, wodgonite {Mn₄(Sn>Ta,Ti,Fe)₄(Ta>Nb)₈O₃₂, microlite

{(Na,Ca)₂Ta₂O₆(O.OH.F)}, pollucite {Cs,Na)(AlSiO₆)H₂0}, lepidolite {(K,Rb) (LiAl)₂ (Al,Si)₄O₁₀ and feldspark

{AlSi₃0₈}.

The richness of the metal content enabled to divide them into two distinct and antagonist groups.

First group contained heavy and toxic metals like vanadium, chromium and to certain extent iron. The

second group contained the beneficial macronutrients like iron, calcium, sodium, potassium,

magnesium and lithium.

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Chapter Two: Experimental

Methodological approach

The reclamation of the lithium mine tailings (LMT) was achieved through five different formulation

mixes, which were referred to as growth media. Moreover, phytostabilization and phytoextraction

processes were achieved through direct sowing of the hyperaccumulant plant seeds in the five

different growth media of four subsamples each. Phytomining of lithium in lieu with heavy metals like

chromium and vanadium was accomplished inside different physiological parts of the hyperaccumulant

plant.

Each growth media had a unique physical or chemical characteristic like pore volume, metal

adsorption capacity, CEC, AEC, salinity, pH, and ORP. The five different formulations were composed of

LMT, homogenized peat and the dewatered municipal biosolids mixed on weight per weight basis (w:

w).

The growth media ingredients were assembled in a sustainable way such that the preference was

given to local and readily available ingredients. The peat was brought from northern Quebec, as well as

the municipal biosolids was obtained from local wastewater treatment plant. Moreover, no major

transport expenses and special handling procedures were needed. The five different growth media

were vigorously tested for their different physical characteristics. The physical characteristics of the

three growth media ingredients were determined separately. First the lithium mine tailing sample was

homogenized, dried, its organic matter content was determined, classified and its texture, pH, salinity

were measured. Likewise for the second and the third ingredients, were peat and municipal biosolids.

In addition, the five different growth media mixes were tested for their pH, ORP, salinity and metal

mass content, through the usage of Perkin Elmer Analyst 100™ atomic absorption spectroscopy (AAS).

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The digestive process was achieved through the addition of concentrated (12N) hydrochloric acid

following the cold extraction procedure. The three ingredients; homogenized peat, lithium mine tailing

and dewatered municipal biosolids were analyzed for the eight metals (V, Cr, Fe, Mg, Na, K, Ca, Li) mass

content, followed by the five different growth media, the organic fertilizer and the leachates that were

generated throughout this research.

As for the metal adsorption and release characteristics of the five distinct growth media, two distinct

experiments were conducted. First the column leachate test, in which one pore volume of DI water

was added specific to each growth media and a pressure of 5 psi units (~35kPa) of pressure was

applied under ambient testing conditions, which generated a leachate. The second experiment was the

Electro-Kinetically metal mass determination. Likewise a one pore volume of DI water was added

specific to each growth media and a DC field of 1 mV∙cm¯¹ was applied on it, which generated a

leachate. Finally the leachates and growth media samples were taken and chemically analyzed for their

metal mass content.

The Brassica juncea ‘the hyperaccumulant’ plant seeds were sown directly on the growth media. They

were germinated, grown, harvested, dried and stored properly. In addition, its leaves, stems, roots and

rhizospheric growth media samples were also analyzed for the eight metals mass content in mg∙kg¯¹ of

dry weight basis. In addition, the monocotyledonous plant seeds were sown on the most favorite

growth media. Finally both of the hyperaccumulant plants were compared based upon their distinct

botanical efficiency parameters.

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Figure 2.1: The methodological flowchart approach ¹

¹: S, the abbreviation for section.

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Figure 2.1: The methodological flowchart approach, cont’d

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Phase one: Formulation of growth media

2.1.1 Lithium mine tailings (LMT) homogenous mixing

The LMT sample was spread on a flat surface in a square manner. The square was divided into eight

equal sections. The mixing was done through the addition of sections 1 on top to 5, 2 to 6, 3 to 7 and 4

to 8 respectively, later on the sections of 1, 5 on top of 3, 7 and 4, 8 on 2 and 6 accordingly. Finally

homogenous mixing was achieved through homogeneous mixing of sections 1, 5 and 3, 7 on top of 2, 6

and 4, 8. [234]

2.1.2 Lithium mine tailings drying process

The lithium mine tailing (LMT) sample was exposed to room temperature until it was thoroughly

dried. Furthermore, its aggregates were crushed and its particle-size analysis was done through the

usage of No.10 (2.00 mm) sieve. (In accordance to ASTM-D421, 2010)

2.1.3 Lithium mine tailings moisture content

This method enabled the laboratory determination of the moisture content on mass basis. The

reduction in mass after drying was regarded as a result of direct evaporation of its moisture content.

Thus a 200 g of LMT sample was dried in an oven at 110 ⁺⁄₋ 5 ˚c for 16 hours until a constant weight

was achieved. The dried sample was removed and weighted directly. Its moisture content was

measured to be 7.008%. Finally the excess samples were stored in a sealed envelope and were kept in

a dry place. (In accordance to ASTM-D2216, 2010)

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2.1.4 Lithium mine tailings organic matter determination

A 25 g of the LMT sample was taken from the oven dried sample. It was placed in a high silica covered

dish inside a muffle furnace. The sample was ashed gradually by increasing the temperature of the

muffle furnace to 440 ˚C. Consequently the sample was ashed for an hour until a constant weight was

obtained to the nearest 0.01 g. Thus the organic carbon content was determined to be 0.29%, which

was multiplied by 1.4, resulted in 0.4% as its total organic matter content.

(In accordance to ASTM-D2974, 2010)

2.1.5 Lithium mine tailings classification

The US standard sieves were arranged on top of each other in a decreasing order such that, the mesh

number 4 (4.76mm), on top of 8 (2.36mm), then 10 (2.00mm) and likewise to the remaining ones, 12

(1.68mm), 16 (1.19mm), 30 (600 μm), 50 (247 μm), 100 (150 μm) and No.200 (75 μm) sieves. After

sieving was achieved the data obtained enabled to construct the “Cumulative grading curve” which

was plotted in figure 2.3. Finally the LMT sample was overwhelmingly classified as sandy (figure 2.2).

(In accordance to ASTM-D2487 and ASTM-D2488, 2010)

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Figure 2.2: Lithium mine tailing (LMT)

2.1.6 Lithium mine tailings texture determination

The LMT sample was passed through the sieve No.4 and retained on sieve number 10 was considered

as coarse sand. Likewise its retained sample on the sieve number 30 was regarded as medium texture

sand and on sieve number 200 as fine textured sandy sample.

The sieves were arranged on top of each other according to their size in a decreasing order such that

the sieve number 4 was on top and the sieve number 200 was on the bottom. A 1000 g of the LMT

sample was passed through the above mentioned sieve complex. The sieving operation was conducted

mechanically by means of lateral and vertical motions, accompanied by jarring action that kept the

LMT samples moving continuously over the surface of the sieve for twenty four minutes. Finally the

mass of the LMT fraction left on each sieve was weighed on the balance to the nearest ± 0.01 g. The

retained mass on each sieve size was summed for accuracy and precision purposes.

(In accordance to ASTM-D422, 2010)

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Figure 2.3: LMT cumulative grading curve

0

20

40

60

80

100

120

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

percentage passing Pe

rce

nt

pas

sin

g

Sieve size (mm)

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2.1.7 Lithium mine tailings pH determination

The test enabled the determination of the H+ ions in LMT solution. It was the determination of the

degree of acidity of the LMT sample, suspended in water and in a 0.01 M calcium chloride solution.

Measurements in both liquids were essential to constitute the full status of the LMT sample. The pH

measurement of the LMT in both water and calcium chloride suspensions were done using the Hach™

electrode. The pH was measured in a calcium chloride solution because calcium displaces some of the

exchangeable aluminum. The low ionic strength counters the dilution effect on the exchange

equilibrium by settling salt. Finally, the pH values obtained in the solution of calcium chloride was

slightly lower, in comparison to the DI water suspension due to the release of more aluminum ions,

which consequently got hydrolysed.

The DI water used had a resistance of 10.0 MΩ cm TC at 11.5°C milli-Q-water, Millipore USA. The

Calcium chloride stock solution (1.0 M) was prepared by dissolving 147 g of Ca Cl₂∙2H₂O in a 1 L of DI

water. The calcium chloride solution (0.01 M) was prepared through the dilution of 20.0 ml of stock 1.0

M CaCl₂ solution in 2 L of water. The pH of this solution ranged between 5 and 7.

The electrodes of the pH meter were placed into the partially settled suspension phase and recorded

its reading. For both solutions, the measurements were taken from an air-dried soil samples that had

been sieved through a No.10 (92 mm) sieve. The pH of the LMT sample was 6.58.

(In accordance to ASTM-D4972, 2010)

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2.1.8 Lithium mine tailings salinity determination

The salinity test revealed the total amount of soluble salts bound on the LMT surface. A 10 g of room

dried LMT sample was taken and added 50 ml of deionized water. The sample solution was shacked on

the BURRELL- Wrist Action™ Pittsburgh, PA-USA vigorous shaker for 5 minutes. The suspended solution

was left to settle for one minute before measuring its salinity. Its salinity was measured through the

salinity meter electrodes suspended in the solution. Finally, the result was rendered to an actual

salinity (ECe) by multiplying the value by a conversion factor based on the values stated in table 2.1.

[235]

The test for salinity was referred to as EC (1:5); which reflected the ratio of one part of LMT sample

added on top of five parts of distilled water in weight per volume basis (wt: v). Finally salinity of 2 dS/m

was considered a low value that does not impede hyperaccumulant plant growth and survival. On the

contrary a value of 6 dS/m was considered an elevated concentration that impedes its growth and

development. The results were tabulated in table 2.1. [235,236]

Table 2.1: EC (1:5) to EC (e) Conversion Factors

Soil Texture

EC (1:5) ‘Multiplication factor’ (dS∙m¯¹) EC (e) (dS∙m¯¹)

Sand 17 0.3638

Sandy loams 13.8

Loams 9.5

Clay loams and light clays 8.6

Medium and heavy clays 7

(Source: Salinity notes, number 8, October 2000)

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2.1.9 Homogenized peat moisture content

The moisture content was expressed as a percent of the oven dry mass. An empty high silica

evaporating dish was weighed under room temperature. A 50.0 g of the homogenized peat (figure 2.4),

sample was weighted. It was oven dried for 1 hour at 105 ⁰C. The oven dried sample was weighed and

recorded its weight to the nearest 0.01 g. For geotechnical purposes the result was referred to as the

moisture content as a percentage value of the oven dried mass, which was measured to be 9 %.

(In accordance to ASTM-D2974, 2010)

Figure 2.4: A homogenized peat sample

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2.1.10 Homogenized peat pH determination

A 3 g of well grounded air dried homogenized peat sample was used. The sample was placed in a 100

ml beaker. A 50 ml of DI water was added in a weigh per volume ratio (w: v). It was occasionally stirred

for 30 min. Afterwards the calibrated Hach™ pH meter electrode was introduced and its pH

measurement was 3.08. (In accordance to ASTM-2976, 2010)

2.1.11 Municipal biosolids pH determination

A 10 ml of freshly obtained municipal biosolids; figure 2.5, mixture was taken. The sample was

centrifuged for 5 min at 3.000 rpm and was left to settle for 10 min. The calibrated Hach™ pH meter

was introduced and its pH reading was recorded as 6.33.

Table 2.2: Biosolids contents

Content Concentration (mg∙l¯¹)

Total and reactive phosphorus 7.52

Phosphorus 8.98

Nitrate 1.08

Chemical oxygen demand 2393

Nitrite 0.732

Volatile acids 4742

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Figure 2.5: Fresh municipal biosolids sample

2.1.12 Growth media pH determination

A 10.0 g of the air dried sample was weighed from of each of the five different growth media sample.

Furthermore, a 10.0 ml of deionized water was added on top of each. The solution was shacked on a

BURRELL- Wrist Action™ Pittsburgh, PA-USA vigorous shaker for 5 min and was left to equilibrate for 1

hour. The calibrated Hach™ pH meter electrode was introduced and its measurements were

summarized in table 2.3.

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2.1.13 ORP (Eh) determination of different GM formulations

2.1.13.1 Introduction

The oxidation reduction potential is an electrical measurement that shows the tendency of a certain

growth media solution to transfer electrons to or from a reference electrode. The measured reading

enabled to estimate whether the five different growth media formulations were aerobic (oxidative

state) or otherwise anaerobic (reductive state). [237]

2.1.13.2 Procedure

The Hach™ pH meter was attached to a platinum based electrode. The instrument was standardized

before usage. A 10.0 g of the growth media sample was placed in a glass container and a 10 ml of DI

water was added. The sample was shaken for 5 min on a BURRELL- Wrist Action™ Pittsburgh, PA-USA

vigorous shaker. The platinum based electrodes were introduced and recorded its reading. Generally

the ORP (Eh) value was represented in milli-volts (mV). It ranges from 800 mV, which reflects an

oxidative state to – 500 mV for a strongly reductive state; the full results were summarized in table 2.3.

[238]

2.1.14 Salinity (EC) levels of different GM formulations

2.1.14.1 Introduction

Metal salts like lithium chloride are serious threat to the environment and ecosystem biodiversity. It

has potential health risks on humans as well as on plant toxicity and loss. As much as 25 % of world’s

cultivated land and more than the half of the irrigated land are affected by salinity, including lithium

chloride salts.

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2.1.14.2 Procedure

A 10 g from each of every five different growth media formulations were taken. A 50 ml of deionized

water was added. The suspension was shaken on a BURRELL- Wrist Action™ Pittsburgh, PA-USA for 5

minutes and was left to rest for a few minutes. The calibrated salinity probe was introduced and

recorded the measurement. It was worth mentioning that an EC range between, 0 to 800 µS∙cm¯¹ was

regarded as suitable for drinking, irrigation and livestock feeding purposes.

On the other hand a range between, 800 to 2500 µS∙cm¯¹ was regarded as a borderline and used

under special management procedures like application of proper drainage and plantation procedures.

[241,242]

Finally, a measurement of EC exceeding 10,000 µS∙cm¯¹ was regarded as non suitable for human

consumption, livestock and for irrigation purposes. Moreover, the total dissolved salts (TDS), was

measured by multiplying it with a constant value of 0.6. Thus the results of the five different growth

media were tabulated in table 2.3.

TDS (mg.L¯¹) = EC ∙ 0.6 [243]

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Table 2.3: ORP, pH, salinity and TDS characteristics per group basis

Group № pH ORP

(mV)

EC

(µS∙cm¯¹)

TDS

(mg∙l¯¹)

Group 1

(100% T) 7.92 -33.4 1264 758.4

Group 2

(50% T, P) 3.74 197.5 538 322.8

Group 3

(33.3% T,P,B) 4.04 170.7 893 535.8

Group 4

(25% T,P, 50% B) 4.64 139.3 728 436.8

Group 5

(50% T and B) 7.16 -5.5 2103 1261.8

2.1.15 Discussion

The different tests described throughout phase one enabled to determine the physical characteristics

of the lithium mine tailing (LMT) like its texture, classification, color, pH, moisture content, ORP and

salinity. The same was achieved for peat and municipal dewatered biosolids, which were the three

main ingredients of the growth media. The research was cornered around the five different growth

media mixes that were formulated and engineered as a means of supporting the hyperaccumulant

plant seed germination and sustainment of growth. Moreover its ability to successfully reclaim the

lithium mine tailings.

First the lithium mine tailings sample was homogenized dried and its initial moisture content was

determined to be 7 %, with a 96% sandy textured sample (table 5.1). In addition, its organic matter

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content was measured to be 0.4 %. Moreover, its pH was 6.58 and was regarded as suitable for plant

germination and survival. Its salinity was 0.363 dS∙m¯¹ which was regarded as non saline, therefore

suitable for the survival of the hyperaccumulant plant seed germination and growth.

The peat sample was one of the ingredients besides the municipal biosolids sample that was amended

to the LMT sample forming the growth media. It was grinded and homogenized its moisture content

was determined to be 9.221% and its pH 3.08. It was considered as an extremely acidic sample, due to

the abundance of organic acids like, humic (HA) and fulvic acids (FA) in its content. [269]

The second ingredient in the growth media was the addition of the dewatered municipal biosolids

sample. It was centrifuged, its pH was measured to be 6.33 and its different characteristics were

summarized in table 2.2.

The different components of the growth media, peat, dewatered municipal biosolids and lithium mine

tailings (LMT) were mixed differently into five different groups. The mixes were expressed in

percentage value, such that the Group 1 had 100% (LMT) which was regarded as the control. The

Group 2 had 50 % (LMT) and 50 % homogenized peat, the Group 3 had 33.3 % of (LMT), homogenized

peat and dewatered biosolids. The Group 4 had 25 % of (LMT) and homogenized peat and 50%

dewatered biosolids and finally the Group 5 had 50 % (LMT) and 50 % dewatered biosolids. Moreover

each of the five different growth media formulations pH, ORP, EC as well as their total dissolved salts

were measured and summarized in table 2.3. The TDS content in each growth media was considered

suitable, for hyperaccumulant plant seed germination and subsequent growth and development

despite its relatively elevated contents in Groups one and five.

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Phase two: Chemical analysis of growth media

2.2.1 Introduction

The chemical analysis of the three growth media ingredients peat, dewatered biosolids and lithium

mine tailing were analyzed for the eight metals; lithium, magnesium, iron, calcium, sodium, chromium,

potassium and vanadium mass content. The extraction process was achieved through the

implementation of the cold extraction procedure. The usage of concentrated (12N) hydrochloric acid

was added on peat dewatered municipal biosolids and lithium mine tailing. They were subjected to

concentrated hydrochloric acid digestion. Later on the samples were shaken on a BURRELL- Wrist

Action™ Pittsburgh, PA-USA wrist action shaker. Then filtered, subsequently diluted and aspired on

Perkin Elmer’s Analyst 100™ flame AAS machine.

Finally the results were tabulated in table 2.4 and 2.5 as well as the content of the organic, ESSF™

Earth Solutions organic fertilizer.

2.2.2 Atomic absorption spectroscopy (AAS)

The flame AAS analysis was used. It is based on the principle of atoms at ground state energy status,

absorbs electromagnetic radiation at specific wavelengths. The radiation was generated from the

hollow cathode lamp. The photons pass through the flame containing atoms of the element. The

degree of absorption is proportional to the concentration of the element in the flame. Therefore the

concentration of the metal was determined in mg∙l¯¹. [245]

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Chemical interferences were corrected through the addition of the proper chemical. It was usually in

a salt form that was added in equal proportions mixed with the standard and the prepared solution

samples [244].

Certified primary elemental standards were used that reduced technical workload and errors. The

metallic element aspiration was done through flame atomic absorption spectroscopy, using the Perkin

Elmer’s, Analyst 100 ™ machine. The main parts of the flame AAS were the hollow cathode lamp, the

nebulizer, the burner system and the radiant detection system [245]. The sample was nebulised on the

spray which was injected into the flame where it was volatilized. Moreover, an atomic gas was

produced into the flame through which its absorbance was measured.

The atomisation was the critical step of atomic absorption. The flame generated was executed under

optimum conditions, depending on each of the specific metallic species specificity. The temperature of

the flame was adjusted accordingly, which was done by using the appropriate fuel/oxidant

combination. The light beam had passed from a hollow cathode lamp onto a mono-chromator and

ended up on a detector that measured the amount of light absorbed. Each and every of the eight

metals had a specific wavelength and operational guidelines.

Finally the light energy was absorbed by the flame through the detector. In addition the amplifier

measured the concentration of that metal in the prepared sample solution. [244,245]

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2.2.3 Metal extraction procedure from LMT, peat and de-B samples and results

The process was achieved through the usage of the cold extraction procedure, or the addition of

concentrated (12N) hydrochloric acid.

2.2.3.1 Hydrochloric acid extraction procedure for eight metals and results

A 2 g oven dried samples of lithium mine tailings and homogenized peat samples were taken. A 20 ml

of concentrated hydrochloric acid, was added with a solid to solution ratio of 1:10 (m: v) [246].

The suspended solution was shaken on a BURRELL- Wrist Action™ Pittsburgh, PA-USA wrist action

shaker for 1 hour and was left overnight to settle [247, 248]. The suspended solution was centrifuged

at 2.000 rpm, for 10 min and was filtered through Whatman No.1 filter paper (particle retention of 11

µm) [247]. Finally the extraction procedure was repeated twice on the above mentioned samples. The

supernatants were then taken and the precipitates were stored. [248]

2.2.4 Lithium measurement

The optimum concentration range was 22 mg∙l¯¹ using a wavelength of 670.8 nm and a sensitivity of

0.035 mg∙l¯¹, fuel Acetylene and oxidant air. The Standard solution was prepared by dissolving 5.324 g

of lithium carbonate (Li₂CO₃) in a minimum volume of (1+1) HCL and diluted to 1 L with deionised

water. 1 ml = 1.00 mg Li (1000mg∙l¯¹). Furthermore, the dilution of the stock lithium solution was

prepared and used as calibration standards at the time of the experimental analysis. The calibration

standards were prepared using the same type of acid and the concentration as that of sample being

analysed. For the analytical purposes, the manufacturer’s manual and recommendations were

followed.

2.2.5 Magnesium measurement

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The optimum concentration range was 8.5 mg∙l¯¹, with a wavelength of 285.2 nm, sensitivity of 0.007

mg∙l¯¹, and detection limit of 0.001 mg∙l¯¹. The fuels used were acetylene and oxidant air. The standard

solution was prepared from dissolving 0.829 g of magnesium oxide (MgO) in 10 ml of redistilled HNO₃

and was diluted to 1 l mark, using deionised or distilled water, such that 1 mL = 0.5 mg of Mg (500

mg∙l¯¹). A 0.1% of Lanthanum chloride was added to samples and standards to overcome the

interference of aluminum, titanium, silica and phosphorus on magnesia signal. Furthermore, the

dilutions of the magnesium solution stock solution were prepared and were used as calibration

standards at the time of the experimental analysis. The calibration standards were prepared using the

same type of acid at the time of concentration as that of sample that was being analysed. For the

analytical purposes, the manufacturer’s manual and recommendations were followed.

2.2.6 Iron measurement

The optimum concentration range was 30 mg∙l¯¹ with a wavelength of 248.3 nm, sensitivity of 0.12

mg/l and detection limit of 0.03 mg∙l¯¹. The fuel used was acetylene and oxidant air. The standard

solution was prepared by mixing 1000 g of pure iron wire dissolved in a 5 ml of redistilled hydrochloric

acid and was made up to 1 L mark with deionised water. 1 ml = 1 mg Fe (1000 mg/l). The stock solution

was used as calibration standards with the prepared dilutions at the time of the experimental analysis.

The calibration standards were prepared using the same type of acid and at the time of concentration

as that of sample being analysed. Finally 0.2% calcium chloride was added on the samples and

standards solutions to overcome the interferences of silica, cobalt, copper and nickel. For the analytical

purposes, the manufacturer’s manual and recommendations were followed.

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2.2.7 Calcium measurement

The optimum concentration range was 17 mg.l¯¹, with the wavelength of 422.7 nm, sensitivity of 0.08

mg/l and detection limit of 0.01 mg/l. The fuel used was acetylene and oxidant air. The stock solution

was prepared in hydrochloric acid and used in standards. A 0.1% potassium chloride was added to the

samples and standards equally to overcome the interferences of Al, Be, P, Si, Ti, V and Zr. Finally for the

analytical purposes, the manufacturer’s manual and recommendations were followed.

2.2.8 Sodium measurement

The optimum concentration range was 6.5 mg.l¯¹, with the wavelength of 589.6 nm, sensitivity of

0.015 mg/l and a detection limit of 0.002 mg/l. The fuel used was acetylene and oxidant air. A 0.1% of

potassium chloride was added to the samples and standards equally to overcome the interferences of

high concentration of mineral acids. For the analytical purposes, the manufacturer’s manual and

recommendations were followed.

2.2.9 Chromium measurement

The optimum concentration range was 9 mg∙l¯¹, at the wavelength 357.9 nm. Sensitivity limit of 0.25

mg/l and the detection limit of 0.05 mg/l. The fuel used was acetylene. The stock solution was

prepared by dissolving 1.923 gr of chromium trioxide (CrO₃) in deionised water. The solution was

acidified with HCL acid and diluted to 1 L mark with deionised water such that 1 ml = 1 mg of Cr (1000

mg/l). A 2% ammonium chloride was added to the samples and the standards equally to overcome the

interferences of iron and excess phosphates that might have been present. For the analytical purposes,

the manufacturer’s manual and recommendations were followed.

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2.2.10 Potassium measurement

The optimum concentration was 22 mg∙l¯¹, at a wavelength of 766.5 nm, sensitivity of 0.04 mg/l and

the detection limit was 0.01 mg∙l¯¹. The stock solution was prepared by dissolving 0.1907g of KCL in

hydrochloric acid and diluted in deionised water to the 1 litre mark. 1 ml = 0.10 mg K (100 mg/l).

Finally a 0.1% of lanthanum chloride was added to the samples and the standards to depress the

interference of mineral acids that might have been present. For the analytical purposes, the

manufacturer’s manual and recommendations were followed.

2.2.11 Vanadium measurement

The optimum concentration was 250 mg∙l¯¹, at the wavelength of 318.4 nm, with a sensitivity of 0.8

mg/l and a detection limit of 0.2 mg/l. The preparation of the stock solution was done through

dissolving 1.7854 gr of vanadium pentoxide V₂O₅, in 10 ml of concentrated in hydrochloric acid. It was

diluted to 1 litre level with deionised distilled water additions. 1 ml = 1 mg V (1000 mg/l). Finally a 0.1%

of potassium chloride was added to the samples as well as the standards to depress the interferences

of Fe, Al, Ti and H₃PO₄. For the analytical purposes, the manufacturer’s manual and recommendations

were followed.

(In accordance with ASTM-E885 and D1971, 2010)

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Table 2.4: Metal mass content in peat, de-B, LMT and organic fertilizer ¹¯²¯³

Peat ²

Mean ± SD

De-B ³

Mean ± SD

LMT ²

Mean ± SD

Organic Fertilizer ³

Mean ± SD

V 17.043

± 0.516

33.89

± 0.774

16.967

± 0.305

5.147

± 0.478

Li <LLD <LLD <LLD <LLD

Cr 0.180

± 0.031

0.361

± 0.041

0.171

± 0.047

0.054

± 0.018

Fe 0.475

± 0.005

1.064

± 0.048

0.494

± 0.005

0.148

± 0.034

Mg 0.76

± 0.745

1.387

± 0.710

0.009

± 0.003

0.327

± 1.744

Ca 0.446

± 0.064

0.342

± 0.008

0.266

± 0.014

0.082

± 0.006

Na 0.047

± 0.002

0.095

± 0.002

0.009

± 0.003

0.014

± 0.001

K NIL

± 0.002

NIL

± 0.001

0.009

± 0.001

30.999

± 30.041

¹: In hydrochloric acid matrix

²: Metals mass content in mg per g of peat and LMT taken assuming uniformity

³: Metals mass content in mg

<LLD: Lower level of detection

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a) The peat

b) The dewatered municipal biosolids

17.043

0 0.18

0.475 0.76 0.446 0.047 0

V

Li

Cr

Fe

Mg

Ca

Na

K

33.89

0 0.361

1.064 1.387 0.342 0.095

0 V

Li

Cr

Fe

Mg

Ca

Na

K

(mg∙g¯¹)

(mg)

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c) The lithium mine tailing

d) The organic fertilizer

Figure 2.6: Metal mass content in a) peat, b) de-MB, c) LMT and d) organic fertilizer

16.967

0 0.171

0.494 0.0095

0.266

0.047

0.009

V

Li

Cr

Fe

Mg

Ca

Na

K

5.1471 <LLD

0.05415

0.1482 0.32775

0.08265

0.01425

30.999

V

Li

Cr

Fe

Mg

Ca

Na

K

(mg)

(mg)

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Table 2.5: Initial metal concentration in five different growth media ¹¯²¯³

Group 1 (100% T) Mean ± SD

Group 2 (50% T and P)

Mean ± SD

Group 3 (33.3% T,P and B)

Mean ± SD

Group 4 (25% T,P and 50% B)

Mean ± SD

Group 5 (50% T and B)

Mean ± SD

V 17.024 ± 0.168

17.176 ± 0.567

16.986 ± 0.359

17.005 ± 0.044

17.043 ± 0.391

Li 0.361 ± 0.191

0.418 ± 0.248

0.247 ± 0.651

0.380 ± 0.358

0.342 ± 0.181

Cr 0.171 ± 0.037

0.171 ± 0.007

0.171 ± 0.013

0.171 ± 0.031

0.171 ± 0.014

Fe 0.475 ± 0.069

0.465 ± 0.066

0.465 ± 0.059

0.465 ± 0.049

0.484 ± 0.034

Mg 0.019 ± 0.003

0.589 ± 1.292

0.484 ± 0.312

0.427 ± 0.287

0.152 ± 0.049

Ca 0.285 ± 0.009

0.408 ± 0.003

0.180 ± 0.007

0.142 ± 0.004

0.142 ± 0.020

Na 0.047 ± 0.003

0.038 ± 0.000

0.047 ± 0.000

0.047 ± 0.002

0.047 ± 0.001

K 0.009 ± 0.013

0.009 ± 0.003

0.009 ± 0.004

0.019 ± 0.022

0.009 ± 0.010

¹: In concentrated hydrochloric acid digestion matrix.

²: Metal mass content in mg∙g¯¹ homogenized samples taken, assuming uniformity.

³: Per different group basis.

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2.2.12 Discussion

The chemical analysis of the three ingredients of the growth media was done through the addition of

the concentrated (12N) HCl acid. It enabled the measurement of the eight different metals mass

content as well as the organic fertilizer. The flame AAS procedure verified the presence of elevated

amounts of vanadium and chromium inside the peat and the lithium mine tailing samples (table 2.4

and figure 2.6).

According to Shotyk [270], peat belongs to the Sphagnum plant family. It has an organic origin

composed of partially decomposed mosses, bryophytes, sedges, grasses, trees, etc. Peat is usually

brownish to blackish in color and it is very susceptible to decomposition and form change with time,

but slowly due to the presence of high concentration of lignin that withstand decay.

When it is taken out of its biosphere and used in horticultural and reclamation purposes, its rate of

decomposition accelerates due to the increase oxygen exposure. As a result its redox conditions are

changed favoring the enhancement of growth and flourishing of the microbial colonies numbers, which

use it as a major nutritional source for their growth and reproduction. [269,271].

Natural peat has a low mechanical strength and high affinity to water [272]. As a result it was

primarily incorporated into the growth media Groups of 2, 3 and 4.

According to Spedding [273], he defined peat as the first stage of coal formation. However, under

increased pressure and temperature it eventually forms coal. The whole process takes over forty

million years. Therefore peat is formed in ecosystems with poor nutrient availability like in waterlogged

areas. Under anoxic conditions, the microbial activity is at its minimum due to the presence of humic

acid (HA), fulvic acid (FA), cellulose based and low molecular weight compounds. These are generally

amphipathic in nature especially in acidic entourage, which increases the availability of heavy metals

like V, Cr, Cd and Fe. These metals might also be bound to the above mentioned compounds forming

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stable organo-mineral complexes due to their chemical and biological bond stability [274,275,276].

These organo metallic complexes hinder heavy metals percolation in ecosystems. However, the HA is

widely present in peat. It is characterized by oxygen containing functional groups like COOH, phenolic,

alcoholic (OH), ketonic and quinonoid (C=O) endings. According to Kendorff and Schnitzer [277] the

humic acid successfully interacts with inorganic minerals forming stable compounds.

According to Chaney and Hundemann [278], Coupal and Lalancette [279], the polar characteristics of

the peat enables it to bind and successfully adsorb and retain organic molecules as well as inorganic

metals like Cd, Zn, Pb, Cu, Hg, Fe, Ni, Cr (VI), Cr (III), Ag and Sb.

According to Crist et al. [280], verified that numerous ion exchange mechanisms takes place on the

peat surface, due to the presence of natural acids like the HA and the FA. It reacts rapidly with heavy

metals, releasing protons at acidic pH, which enables the displacement of metals by others.

According to Wolf et al. [281], Ong and Swanson [282] succeeded proving that increasing calcium

concentration in peat enhanced the sorption of heavy metals like Pb, Cd, Cu and Zn. As an outcome

different peat surface adsorption mechanisms occurs between the positively charged metals and the

negatively charged radicals, forming bonds and stable entities, that eventually increases its surface

area. Moreover, Chen et al. [283], proved after frequent additions of Cu (NO₃)₂ under certain pH

conditions, adsorption-complexation interactions occurred between the cations and the anion entities.

Sharma and Forster [284] were able to prove the strong sorption of peat surface which was possible in

acidic pH. Thus the Cr (VI) on the peat surface was strongly adsorbed but only a negligible amount was

released of the total Cr (VI). It was verified that the Group 4 growth media, which was composed of

25% LMT, 25% homogenized peat and 50 % of dewatered municipal biosolids at a pH of 4.64. The

initially measured chromium content was 0.171 mg∙gr¯¹. However, the hyperaccumulant plant Brassica

juncea, managed to phytoextract from the Group 4 growth media and translocate 0.0655 mg in its

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roots 0.296 mg in its stem, 0.1595 mg in its leaves and phyto-stabilize 0.256 mg∙gr¯¹ around its

rhizosphere, totaling 0.5212 mg∙gr¯¹. Comparing the two totals revealed a considerable difference

between them, which is a clear indication that the above mentioned sorption processes had surely

occurred. It was speculated that the roots of the hyperaccumulant plant was able to extract the hard

bound chromium from the growth media of the Group 4.

Finally as a source for the elevated concentrations of vanadium 17.043 mg∙gr¯¹, chromium 0.1805

mg∙gr¯¹ and iron 0.475 mg∙gr¯¹ in the peat content, as measured in table 2.4 and figure 2.6, was

speculated to two main sources.

The first source was speculated to be the direct result of the parent material formation. Moreover,

the second source might be due to the different anthropogenic and industrial activities that might have

occurred or still occurring near or around its vicinity. Like fossil fuel combustion or coal burning in

which the content of vanadium in fuel might range between 60 to 1000 mg∙kg¯¹ and from 2 to 100

mg∙kg ¯¹ in coal combustion. [285,286]

The purpose behind the usage of dewatered municipal biosolids was to enhance in the different

growth media Groups with a potential source of viable microorganism colonies for different symbiotic

interactions that might occur with the hyperaccumulant plant root system. Eventually this interactions

will enhance the nutrient availability specially nitrogen and phosphorus to the hyperaccumulant plant

uptake. It will also render the hard bound metals, like vanadium, chromium, iron and lithium more

accessible to plant uptake translocation and storage into its different physiological parts. [287]

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Chapter three: Column and leachate test

3.3.1 Introduction

The column leaching procedure is a standard method for generating aqueous leachate from the five

different growth media formulations. A 0.3 g of lithium chloride was amended in every 100 g of growth

media mix. Each growth media was placed inside the column (figure 3.7). A one pore volume of

deionized water was added specific to each group. Furthermore, a 5 psi (~ 35kPa) of pressure was

exerted on the column, which resulted in leachate generation. It provided the proper means to analyse

the solute metal mass content solubility in the solvent. The effluent collected was aspired by the flame

atomic absorption spectroscopy (AAS). It enabled the measurement of the lithium and the remaining

seven metals mass content. The column leachate procedure enabled to conclude the metal absorbing

and releasing abilities of the five different growth media formulations for the eight metallic elements.

The maximum permissible particle size was 10mm; larger particles were removed through sieving that

ensured adequate compaction. Organic immiscible materials were removed from the column since it

might have lead to plugging and fouling. Volatilization was a big problem to column test procedure. Its

effect was negligible due to the implementation of proper handling and testing procedures.

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3.3.2 General assembly

Each of the circular cylindrical sections of the column apparatus were thoroughly washed, cleaned

and consequently dried. After each repetitive experiment they were placed properly on top of each

other, starting from the bottom cap, the middle cylindrical rings and ending up with the upper cap.

They were fitted and fastened with outer screws, bolts and metallic rings. Throughout the column test

experiment, the applied pressure was synchronized by the pressure tower, alongside the column

apparatus. It regulated the inflow of air pressure that was exerted on the deionized water. The applied

pressure was able to wash the different growth media mix profiles and dissolve the solute within the

solvent effluent.

3.3.3 Peat homogenization process

A 180 g of the peat sample was taken; its lumps and formations were crushed with a spatula.

Furthermore the sample was homogenized with a blender. It was covered with an opaque cap to

deprive it for absorbing moisture and light penetration from its surrounding.

3.3.4 Municipal biosolids dewatering procedure

A 250 ml of raw sludge or municipal biosolids suspension was taken. It was centrifuged for 5 minutes

at 3,000 rpm speed. The supernatant was decanted and the concentrate was used as an ingredient in

the growth media formulations.

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3.3.5 LiCl amending procedure

The five different growth media formulations or groups were amended with a 0.3 g of lithium

chloride, per 100 g of the growth media mix.

3.3.6 Homogenous mixing

The different growth media formulations or Groups were spread on a flat surface in a square manner.

The square was divided into eight equal divisions. Furthermore, the primary mixing was done by the

addition of divisions 1 on top 5, 2 on top 6, 3 on top 7 and 4 on top 8. The process was continued by

mixing the divisions of (1, 5) on top of (3, 7) and (4, 8) on top of (2 and 6) respectively. Finally a

complete homogenization was achieved through mixing divisions (1, 5) and (3, 7) on top of (2, 6) and

(4, 8). [234]

3.3.7 Experimental procedure

The five different growth media mixes were amended with 3 g∙kg¯¹ of LiCl. It was left to react for ten

consecutive days. Furthermore, the five different media formulations were introduced into the column

apparatus one after the completion of the other. The void volume was calculated for each growth

media using the equation stated beneath. Moreover, a one pore volume of deionized water was added

specific to each growth media. In addition, a pressure of 5 psi (~35kPa) was applied on the column

apparatus; as a result it generated effluent.

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Vp = Vc {M/ [(1+W) ∙S ∙ D]}

Where; Vp = void volume in column in cm³

Vc = volume of column in cm³

M= as packed mass of the material including its moisture, contained in the

column in g.

W= moisture content of the material inside the column in g- water/g-solids.

S= Specific gravity of the material, unit less.

D= density of water in g/cm³.

The degree of saturation was determined through the calculation of porosity (n) by using the following

relationship: n = Vp/Vc

(In accordance with ASTM-D4874, 2010)

The column apparatus was filled with a unique growth medium mix. A pressure of 5 psi units (~35

kPa) was adjusted and exerted by the pressure tower. Furthermore, the same growth medium profile

was washed for three consecutive times. Every wash consisted one pore volume of deionized water

specific to each growth media. In addition, the column and the pressure tower were run under

ambient temperature and under normal testing protocols of room temperature 22 °C and humidity 40

%. The collected leachate samples were stored and refrigerated (at 7˚C) in sealed containers.

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3.3.8 Leachate digestion procedure in concentrated hydrochloric acid

The collected leachate samples were tested for their salinity, pH and oxidation reduction potential.

Moreover, a 3 ml of the generated leachate was digested with 3 ml of concentrated (12N) hydrochloric

acid. The digestion was left to proceed for 48 hrs. Its metal mass content was analyzed through the

atomic absorption spectrometry (AAS). Finally the concentrations of the eight metallic elements were

calculated using the following formula;

Metallic element concentration (mg/l) = (C) ∙ (d.f.)

Where;

C= concentration of element in sample solution in ml.

df = is the dilution factor.

3.3.9 Organic fertilizer analysis

A 3 ml of the organic fertilizer was taken. It was digested by the addition of 3 ml concentrated (12N)

hydrochloric acid on volume per volume basis (v: v). The digestion process was proceeded for 72 hrs

and its metal mass content was analyzed through the flame atomic absorption spectrometry (AAS).

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2.3.10 Metal mass content analysis per group basis and results

A 10 g from different growth media formulations was taken. Furthermore, the samples were oven

dried at 105 ±5°C for one hour. Their moisture content was measured and was each sample was

digested in a 100 ml of (12N) concentrated hydrochloric acid.

Finally the eight metallic element concentrations were measured using the flame AAS. The results

were summarized in tables 3.1, 3.2 and 3.3.

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Table 3.1: Leachate characteristics per different group and wash basis ¹¯²

Group №

Washes №

pH ORP (mV)

EC (mS∙cm¯¹)

1

100% lithium mine tailing

(LMT)

1 7.38 -52.8 11.99

2 8.12 -60.7 5.79

3 8.26 -68.9 2.53

2

50% (LMT) and peat (P)

1 7.29 -13.2 0.662

2 7.29 -13.0 0.531

3 7.45 -22.4 0.390

3

33.3% (LMT),(P) and

biosolids (B)

1 4.14 167.1 5.80

2 4.14 166.5 3.36

3 4.09 169.1 2.030

4

25% (LMT),(P) and 50%

(B)

1 4.21 163.4 4.69

2 4.43 151.1 2.171

3 4.77 131.4 1.704

5

50% (LMT) and (B)

1 6.50 32.4 7.13

2 6.83 13.2 3.21

3 7.03 1.9 1.840

¹: The EC was measured at 21 °c non linear NaCl

²: LMT stands for lithium mine tailing, P stands for homogenized peat and B stands for dewatered

biosolids

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a) pH

b) ORP

0

1

2

3

4

5

6

7

8

9

pH

(pH)

(Group №-Wash №)

-100

-50

0

50

100

150

200

G1-W1

G1-W2

G1-W3

G2-W1

G2-W2

G2-W3

G3-W1

G3-W2

G3- W3

G4- W1

G4- W2

G4-W3

G5-W1

G5-W2

G5-W3

ORP (mV) (mV)

(Group №-Wash №)

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c) EC

Figure 3.1: pH, ORP and EC per group and wash basis ¹

¹: The leachate generated after three consecutive washes

0

2

4

6

8

10

12

14

EC (mS.cm¯¹) (m

S.cm

¯¹)

(Group №-Wash №)

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Table 3.2: Leachate metal mass content per Group and wash basis ¹

GM

Wash №

V Mean ±

SD

Li Mean ±

SD

Cr Mean ±

SD

Fe Mean ± SD

Mg Mean ±

SD

Ca Mean ±

SD

Na Mean ±

SD

K Mean ±

SD

1 (100% T)

1 67.719 ±0.668

12.632 ±1.001

0.726 ±0.031

1.912 ±0.049

0.114 ±0.004

0.612 ±0.010

0.191 ±0.004

0.038 ±0.006

2 68.249 ±0.485

6.733 ±0.095

0.726 ±0.025

1.874 ±0.044

0.114 ±0.005

0.573 ±0.006

0.191 ±0.003

0.038 ±0.009

3 67.867 ±0.293

1.606 ±0.748

0.726 ±0.016

1.874 ±0.018

0.114 ±0.001

0.573 ±0.027

0.191 ±0.000

0.267 ±0.913

2 (50% T and

P)

1 100.66 ±0.090

<LLD 1.014 ±0.032

2.761 ±0.021

0.169 ±0.006

0.619 ±0.018

0.2818 ±0.001

0.056 ±0.003

2 100.19 ±0.178

<LLD 1.07

±0.016 2.761 ±0.146

0.225 ±0.007

0.676 ±0.032

0.2818 ±0.001

0.056 ±0.008

3 100.21 ±0.684

<LLD 1.07

±0.012 2.761 ±0.028

0.225 ±0.007

0.619 ±0.012

0.2818 ±0.002

0.056 ±0.003

3 (33.3% T,P and B)

1 176.61 ±0.480

<LLD 1.789 ±0.039

4.871 ±0.095

3.181 ±0.184

1.59 ±0.021

0.497 ±0.001

0.894 ±0.161

2 177.40 ±0.678

<LLD 1.789 ±0.037

4.871 ±0.027

1.491 ±0.035

0.894 ±0.009

0.497 ±0.002

0.596 ±0.213

3 177.20 ±0.611

<LLD 1.789 ±0.009

4.871 ±0.016

0.795 ±0.007

1.506 ±0.016

0.497 ±0.003

0.198 ±0.045

4 (25% T,P and 50% B)

1 253.90 ± 0.075

<LLD 2.576 ±0.019

7.013 ±0.005

2.862 ±0.019

2.576 ±0.011

0.715 ±0.001

2.29 ±0.003

2 255.91 ±0.228

<LLD 2.576 ±0.043

7.013 ±0.043

1.288 ±0.020

1.145 ±0.006

0.715 ±0.002

0.429 ±0.017

3 255.05 ±0.519

<LLD 2.576 ±0.019

7.013 ±0.054

1.001 ±0.016

1.431 ±0.018

0.715 ±0.002

6.154 ±1.868

5 (50%

T, and B)

1 199.19 ±0.356

22.651 ±0.241

2.109 ±0.023

5.44 ±0.089

1.221 ±0.008

3.997 ±0.008

0.555 ±0.001

0.222 ±0.014

2 198.08 ±0.514

7.550 ±0.113

1.998 ±0.047

5.44 ±0.099

0.666 ±0.004

1.887 ±0.013

0.555 ±0.001

0.111 ±0.004

3 199.198 ±0.413

3.775 ±0.002

1.998 ±0.062

5.329 ±0.033

0.555 ±0.011

1.554 ±0.023

0.555 ±0.001

0.111 ±0.007

¹: Metallic element concentration in mg

<LLD: Lower level of detection

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Figure 3.2: Variation of metal mass content per Group and wash basis ¹¯²¯³

¹: In concentrated hydrochloric acid matrix

²: Metal mass content in mg ³: The vanadium concentrations were capped at 5 mg, below are its actual values

G1-W1 G1-W2 G1-W3 G2-W1 G2-W2 G2-W3 G3-W1 G3-W2 G3-W3 G4-W1 G4-W2 G4-W3 G5-W1 G5-W2 G5-W3

67.714 68.249 67.869 100.66 100.19 100.21 176.612 177.4 177.2 253.9 255.91 255.05 199.19 198.08 199.19

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Cr Mg Ca Na (mg)

Group № - Wash №

0

5

10

15

20

25

Li Fe V K (mg)

Group № - Wash №

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Table 3.3: Metal mass left in Groups after CWP ¹¯²

Group 1

(100% T)

Mean ± SD

Group 2

(50% T and P)

Mean ± SD

Group 3

(33.3% T,P and B)

Mean ± SD

Group 4

(25% T,P and 50% B)

Mean ± SD

Group 5

(50% T and B)

Mean ± SD

V 17.024

± 0.483

17.024

± 0.166

17.119

± 0.070

16.986

± 0.253

17.119

± 0.894

Li <LLD 0.304

± 0.122 <LLD <LLD

0.019

± 0.004

Cr 0.171

± 0.021

0.171

± 0.043

0.171

± 0.031

0.171

± 0.036

0.171

± 0.008

Fe 0.475

± 0.044

0.465

± 0.046

0.465

± 0.026

0.465

± 0.031

0.475

± 0.046

Mg 0.001

± 0.003

0.465

± 0.413

0.161

± 0.047

0.142

± 0.012

0.171

± 0.039

Ca 0.266

± 0.021

0.427

± 0.032

0.123

± 0.014

0.133

± 0.013

0.133

± 0.008

Na 0.047

± 0.001

0.0475

± 0.002

0.047

± 0.001

0.047

± 0.004

0.047

± 0.002

K NIL

± 0.002

0.0095

± 0.004

0.0095

± 0.005

0.0095

± 0.007

0.009

± 0.003

¹: In concentrated hydrochloric acid matrix

²: Metal mass in mg∙g¯¹, assuming uniformity

<LLD: Lower level of detection

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Figure 3.3: Metal mass left in Groups after column washing procedure ¹¯²

¹: Vanadium concentration was capped at 0.2 mg, below are its actual values

²: Metals mass content in mg∙g¯¹, assuming uniformity

Group 1 Group 2 Group 3 Group 4 Group 5

V (mg.) 17.024 17.024 17.119 16.986 17.119

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

V Li Cr Fe Mg Ca Na K

Group 1 Group 2 Group 3 Group 4 Group 5

(mg)

(Metals)

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Figure 3.4: Initial metal mass content in different Groups ¹¯²¯³

¹ Vanadium, mass content was capped at 0.2 mg below are its real

²: Metals mass content in mg∙g¯¹, assuming uniformity

³: Prior to CWP

Group 1 Group 2 Group 3 Group 4 Group 5

V(mg) 17.024 17.176 16.986 17.005 17.043

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

V Li Cr Fe Mg Ca Na K

Group 1 Group 2 Group 3 Group 4 Group 5

(mg)

(Metals)

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Figure 3.5: Metal mass removed ¹

¹: In percentage basis

0

0.5

1

1.5

2

2.5

3

Cr Fe Mg Ca Na V

Group-1 Group-2 Group-3 Group-4 Group-5

% R

em

ove

d a

fte

r C

WP

(Metals)

0

5

10

15

20

25

30

35

Group-1 Group-2 Group-3 Group-4 Group-5

li k

% r

emo

ved

aft

er

CW

P

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a) Lithium

b) Vanadium

Figure 3.6: Initial vs. left vs. leachate of metal mass content per group basis

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6

Li (Initial) Li (Leachate) Li (Left) (mg)

0

100

200

300

400

500

600

700

800

900

0 1 2 3 4 5 6

V (Initial) V (Leachate) V (Left) (mg)

Group №

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c) Chromium

d) Iron

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6

Cr (Initial) Cr (Leachate) Cr (Left) (mg)

0

5

10

15

20

25

0 1 2 3 4 5 6

Fe (Initial) Fe (Leachate) Fe (Left) (mg)

Group №

Group №

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e) Magnesium

f) Calcium

0

1

2

3

4

5

6

0 1 2 3 4 5 6

Mg (Initial) Mg (Leachate) Mg (Left)

(mg)

Group №

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

Ca (Initial) Ca (Leachate) Ca (Left) (mg)

Group №

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g) Sodium

h) Potassium

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6

Na (Initial) Na (Leachate) Na (Left) (mg)

Group №

0

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6

K (Initial) K (Leachate) K (Left) (mg)

Group №

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Figure: 3.7: Column apparatus (front vs. aerial views)

Where; the “A” is the DI water compartment and the “B” is the GM compartment

The Top Cap

The Bottom Cap

Effluent (leachate generated) tube

Pressure regulator

gauge

The Pressurized

air inflow tube.

tube

Fastening

screws

A

B

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2.3.11 Discussion

The column wash process (CWP - figure 3.7) enabled to measure the metal holding capacity of the five

different growth media Groups. The generated leachates were digested by concentrated (12N)

hydrochloric acid. The solution was filtered, diluted and was aspired through the Perkin Elmer’s Analyst

100™ flame atomic absorption spectroscopy machine (AAS). Moreover, the total mass balance was

calculated for each metal. The total mass was rendered in milligram by multiplying its concentration

(ppm) by its leachate volume [261, 262], the full results were summarized in table 3.2.

The pH, ORP and the EC of the generated leachate was measured after consecutive wash. The results

were summarized in table 3.1 and figure 3.1. The decreasing trend of ORP and EC values after each

wash was assumed to be due to the decrease concentration of soluble salt ions like sodium or chlorine

in the growth media. As well as the decrease of oxygen content due to the prevalence of anoxic or

waterlogging condition (figure 3.1-b & c).

As for the slight pH increase after each consecutive wash was assumed to be due to the increased

presence of water molecules.

It was observed that lithium was strongly adsorbed in acidic growth media Groups of 2, 3 and 4,

rather than vanadium, chromium and the rest of the metals which were easily leached. On the

contrary lithium was easily leached under neutral pH conditions like in Groups 1 and 5, likewise, to the

remaining metals. Finally, the strong adsorption of lithium in Groups 2, 3 and 4 was assumed to be due

to the abundance of peat ingredient in their formulation. The peat in these Groups played the role of a

buffer and a storage reservoir, hindering lithium leachate into the lithosphere.

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Phase four: Impact of electrokinetics (EK) on growth media and

metal content determination

3.4.1 Introduction

The electrokinetic application on growth media (EK-GM) technique was tailored and applied according

to the outcome needs. It was a viable alternative approach to the mobilization of the hard bound eight

metals and their mass content determination. They were presumably present in abundance within the

growth media Groups.

The EK-GM process consisted of DC source applied as 1V∙cm¯¹. It was related to the EK cell via inert

electrodes immersed in the growth media (figure 3.9, 3.10). As a result the applied DC generated an

electrical potential gradient, which via the electrodes enabled the migration of the charged ions

enabling electroosmotic, electrophoretic and electromigratory movements to respective electrodes

the anode and cathode. [265, 288, 289, 290]

The electromigratory, electrophoretic and electroosmotic movement was made possible due to the

one pore volume of deionized water supplemented specific to each growth media group. It was

dragged towards the cathode electrode and throughout the course it was dissociated to oxygen and

hydroxide ions. These ions were an important source for electron transport on both electrodes such

that;

H2O → 2H⁺ + ½ O2 (gas) + 2e¯ (at the anode) and

2H2O + 2e¯ → 2OH¯ + H2 (gas) (at the cathode)

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The DC supplied throughout the growth media, might have lead to the formation of two distinct

fronts around each confronting electrodes. The different fronts that were created and the radicals

generated were carried towards the respective electrode resulting in electro-migration,

electrophoresis and electro-osmosis and enhancement of diffusion process. [265, 266, 302]

The generated effluents on both electrodes were collected in PPE bottles. Their pH, ORP, EC and

moisture content was also measured (tables 3.4, 3.7 and figures 3.11, 3.13). The samples were

chemically digested by concentrated (12N) hydrochloric acid. Then they were filtered and diluted.

Finally the samples were aspired and the eight metals mass content was determined (tables 3.5, 3.6

and figure 3.12).

3.4.2 Electrokinetics cell description

The experimental cell was made from a transparent Plexiglas material of 24 cm by 8 cm and 6 cm in

size. Its cap contained 18 stainless steel rods of 1cm apart, figure 3.8.

Figure 3.8: Cap of the electrokinetics cell and the stainless steel rods

The bottom plate had two distinct holes 20 cm apart. It was fastened onto the anode and the

cathode, figure 3.9 through stainless steel screws.

The outer cover and the stainless steel rods.

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102

Figure 3.9: Bottom plate of electrokinetics cell in an upright position

The electrodes were made of stainless steel material. They were hollow inside perforated on its sides

that allowed the generated leachate to percolate (figure 3.9). The low DC field deprived electrode

corrosion and fouling. The growth media was added inside the cell and a one pore volume of deionized

water was added specific to each growth media group. The cell was properly sealed by its cap that

prevented water loss due to evaporation. The whole unit was linked to the DC power supply generator,

figures 3.10.

The bottom side of the E.K cell with its two holes.

The upper side of the E.K cell with its two electrodes in place.

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103

Figure 3.10: Electrokinetics setup and the DC power generator in process

3.4.3 Experimental process and results

The each of the five different growth media groups was amended with 3 gr∙kg¯¹ lithium chloride.

Moreover, one pore volume of deionized water was added specific to each growth media holding

capacity. The DC power generator was turned on for two consecutive days. It was kept on a constant

voltage gradient of 1V∙cm¯¹ of the EK cell. The amperage decreased with time due to the

electroosmotic transport of water towards opposing electrodes. However due to the very low DC field

of 1V.cm¯¹ electrode corrosion and fouling was not observed. Moreover the electric power

consumption was calculated by multiplying the amps with the voltage divided by a thousand. Thus the

power consumption was recorded around 0.1584 KWh.

The cell with

its cap

Effluent

collecting bottles

The cathode The anode

The metallic stand

The DC power generator The voltage and the Amperage.

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The pH, ORP and salinity characteristics of the collected leachate from the anode and the cathode was

measured and refrigerated at 4°C. Moreover, the growth media in the EK cell was divided into three

distinct parts (or slices). The first slice was around the cathode. The second slice was around the anode

and the third slice was the middle part of the EK cell. Thus representative samples were taken from the

three distinct slices. The samples were oven dried at 105 °C for one hour. Moreover, their pH, ORP and

salinity characteristics were also measured. The eight metal mass concentrations were determined

through the application of cold digestion procedure (12N HCL). The samples were digested and then

diluted with deionized water of resistance 10.0 Ω cm TC at 11.5°C –milli-Q-water, Millipore USA. Finally

the samples were aspired on Perkin Elmer’s Analyst 100™ flame AAS. The full results were summarized

in tables 3.4, 3.5, 3.6 and expressed graphically in figures 3.11, 3.12 and 3.13.

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Table 3.4: pH, ORP and EC in five different groups ¹¯²

Group 1

(100% T)

Group 2

(50% T and P)

Group 3

(33.3% TP and B)

Group 4

(25% TP and 50% B)

Group 5

(50% T and B)

Cathode

(L)

pH 3.34 7.90 5.30 3.28

2.32

ORP 214 -48.1 101.5 217.3 273.1

EC 7.63 0.474 1.474 6.12 29.7

Anode

(L)

pH 11.97 ³ 7.02 8.85 12.33

ORP - 282.2 ³ 2.4 -102.8 -303.0

EC 31.2 ³ 1.396 3.79 17.01

Cathode

(GM)

pH 1.9 3.54 3.51 3.04 3.99

ORP 296.0 202.4 204.2 231.4 176.3

EC 6.61 0.888 1.159 1.124 2.057

Anode

(GM)

pH 11.05 3.49 7.26 8.91 9.66

ORP -228.9 205.3 -11.2 -106.4 -149.5

EC 1.984 0.782 0.235 1.009 0.729

Middle cell

(GM)

pH 9.72 3.53 4.26 4.06 7.82

ORP -152.8 202.7 161.0 172.7 -43.3

EC 0.29

0.923 0.597 0.00014 0.2014

¹ ORP expressed in mV and EC expressed in mS∙cm¯¹

² L, GM are referred to leachate and growth media respectively

³ Absence of sample

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a) pH in per group basis

b) ORP per group basis

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Cathode (G.M.) Anode (G.M.) Middle (G.M.)

pH

Group №

-300

-200

-100

0

100

200

300

400

0 1 2 3 4 5 6

Cathode (G.M.) Anode (G.M.) Middle (G.M.)

(mV)

Group №

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c) EC per group basis

Figure 3.11: pH vs. ORP vs. EC per group basis

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6

Cathode (G.M.) Anode (G.M.) Middle (G.M.)

mS.

cm¯¹

Group №

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Table 3.5: Metal mass content in five different Groups ¹¯²¯³

Li

Mean ± SD

V Mean ±

SD

Fe Mean ±

SD

Cr Mean ±

SD

Mg Mean ±

SD

Ca Mean ±

SD

Na Mean ±

SD

K Mean ±

SD

Group1 (100% LMT)

CL 0.2832 ± 6.175

1.202 ± 0.028

0.041 ± 0.260

0.019 ± 0.030

0.0039 ± 0.005

0.032 ± 0.087

0.0006 ± 0.008

0.665 ± 0.036

AL 0.328 ± 3.005

1.200 ± 0.087

0.030 ± 0.095

0.015 ± 0.049

0.0006 ± 0.001

0.002 ± 0.003

0.0006 ± 0.010

0.021 ± 0.070

C-GM 0.019 ± 0.001

17.157 ± 0.374

<LLD <LLD 0.009 ± 0.001

0.399 ± 0.097

0.0285 ± 0.049

0.066 ± 0.030

A-GM 0.741 ± 0.033

17.062 ± 0.453

0.541 ± 0.281

0.237 ± 0.031

0.019 ± 0.000

0.665 ± 0.057

0.019 ± 0.026

0.104 ± 0.035

M-GM 0.152 ± 0.007

17.195 ± 0.461

0.589 ± 0.617

0.247 ± 0.040

0.019 ± 0.002

0.655 ± 0.119

0.019 ± 0.012

0.095 ± 0.039

Group2 (50% LMT

and P)

CL NIL 6.908 ± 0.146

0.178 ± 0.266

0.091 ± 0.020

0.030 ± 0.014

0.060 ± 0.022

0.003 ± 0.008

0.201 ± 0.397

AL ³

C-GM 0.304 ± 0.018

17.328 ± 0.251

0.456 ± 0.335

0.228 ± 0.032

0.693 ± 0.181

0.855 ± 0.166

0.009 ± 0.010

0.076 ± 0.027

A-GM 0.342 ± 0.020

17.309 ± 0.346

0.446 ± 0.039

0.228 ± 0.055

0.731 ± 0.371

1.035 ± 0.093

0.009 ± 0.004

0.104 ± 0.005

M-GM 0.285 ± 0.012

17.271 ± 0.086

0.446 ± 0.103

0.228 ± 0.024

0.684 ± 0.303

0.836 ± 0.150

0.019 ± 0.016

0.171 ± 0.068

Group3 (33.3% LMT,P and B)

CL 0.029 ± 0.009

2.936 ± 0.223

0.077 ± 0.534

0.0371 ± 0.030

0.016 ± 0.021

0.016 ± 0.011

0.0016 ± 0.009

0.009 ± 0.010

AL 0.034 ± 0.012

3.849 ± 0.124

0.109 ± 0.293

0.052 ± 0.016

0.019 ± 0.013

0.021 ± 0.014

0.002 ± 0.003

0.017 ± 0.025

C-GM 0.247 ± 0.028

17.366 ± 0.713

0.693 ± 0.467

0.304 ± 0.061

0.712 ± 0.694

0.608 ± 0.153

0.009 ± 0.004

0.104 ± 0.034

A-GM 0.418 ± 0.041

17.385 ± 0.687

0.503 ± 0.491

0.228 ± 0.050

0.722 ± 1.044

0.750 ± 0.059

0.0095 ± 0.015

0.104 ± 0.006

M-GM 0.285 ± 0.018

17.404 ± 0.243

0.484 ± 0.223

0.228 ± 0.019

0.760 ± 1.800

0.456 ± 0.021

0.009 ± 0.009

0.085 ± 0.011

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Li

Mean ± SD

V Mean ±

SD

Fe Mean ±

SD

Cr Mean ±

SD

Mg Mean ±

SD

Ca Mean ±

SD

Na Mean ±

SD

K Mean ±

SD

Group4 (25%

LMT,P and 50%

B)

CL 0.1672 ± 0.041

3.686 ± 0.479

0.146 ± 0.359

0.060 ± 0.033

0.062 ± 0.044

0.035 ± 0.074

0.002 ± 0.023

0.064 ± 0.083

AL 0.156 ± 0.053

4.180 ± 0.123

0.111 ± 0.380

0.054 ± 0.024

0.026 ± 0.012

0.016 ± 0.026

0.004 ± 0.020

0.026 ± 0.030

C-GM 0.133 ± 0.001

16.739 ± 0.508

1.035 ± 1.599

0.399 ± 8.105

0.437 ± 0.077

0.617 ± 0.103

0.009 ± 0.005

0.114 ± 0.038

A-GM 0.760 ± 0.063

16.758 ± 0.162

0.456 ± 0.029

0.228 ± 0.032

0.494 ± 0.077

0.494 ± 0.081

0.009 ± 0.003

0.294 ± 0.079

M-GM NIL 16.777 ± 0.302

0.589 ± 0.292

0.237 ± 0.019

0.408 ± 0.138

0.731 ± 0.187

0.009 ± 0.023

0.057 ± 0.035

Group5 (50% LMT

and B)

CL 0.003 ± 0.001

3.006 ± 0.289

<LLD <LLD 0.003 ± 0.003

0.006 ± 0.015

0.011 ± 1.859

0.034 ± 0.066

AL 1.882 ± 1.949

12.937 ± 0.424

0.355 ± 0.251

0.170 ± 0.012

0.029 ± 0.005

0.007 ± 0.010

<LLD 0.800 ± 1.558

C-GM 0.038 ± 0.003

16.758 ± 0.431

<LLD <LLD 0.304 ± 0.020

0.085 ± 0.021

0.009 ± 0.003

0.266 ± 0.024

A-GM 0.304 ± 0.003

16.739 ± 0.116

0.722 ± 0.735

0.228 ± 0.027

0.437 ± 0.084

0.408 ± 0.056

0.009 ± 0.020

0.104 ± 0.033

M-GM 0.057 ± 0.004

16.739 ± 0.186

4.512 ± 7.669

0.228 ± 0.019

0.361 ± 0.093

0.494 ± 0.060

<LLD 0.095 ± 0.010

¹: In hydrochloric acid matrix

²: Metal mass content in mg∙ g¯¹

³: Absence of sample

<LLD: Lower level of detection

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Table 3.6: Metal mass difference between column and EK leachate ¹¯²¯³

Group № Li V Cr Fe Ca Na K Mg

1

(100% LMT)

EK 874 3433.3 50.35 103.55 50.35 1.9 981.35 6.65

CL 313.7 1681.5 18.05 47.5 15.2 4.75 0.95 2.85

2

(50% LMT and

P)

EK NIL 1721.1 22.8 44.65 15.2 0.95 50.35 7.6

CL <LLD 1696.7 17.1 46.55 10.45 4.75 0.95 2.85

3

(33.3% LMT, P

and B)

EK 32.3 3401 44.65 93.1 19 1.9 13.3 18.05

CL <LLD 1689.1 17.1 46.55 15.2 4.75 8.55 30.4

4

(25% LMT, P

and 50% B)

EK 138.7 3347.8 49.4 111.15 22.8 2.85 39.9 38.95

CL <LLD 1685.3 17.1 46.55 17.1 4.75 15.2 19

5

(50% LMT and

B)

EK 243.2 3328.8 21.85 45.6 4.75 6.65 121.6 5.7

CL 193.8 1704.3 18.05 46.55 34.2 4.75 1.9 10.45

¹: Eight metal concentrations in mg∙l¯¹

²: EK and CL abbreviations to electrokinetics and column leachate respectively

³: First wash effluent metal concentration was considered from column leachate procedure

<LLD: Lower level of detection

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Figure 3.12: Metal mass difference between EK vs. column leachate ¹ ¹: Metal concentrations in mg∙l¯¹

0

500

1000

1500

2000

2500

3000

3500

4000

EK CL EK CL EK CL EK CL EK CL

1 2 3 4 5

Li V Fe K (mg∙l¯¹)

Group №

0

10

20

30

40

50

60

EK CL EK CL EK CL EK CL EK CL

1 2 3 4 5

Cr Ca Na Mg (mg∙l¯¹)

Group №

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Table 3.7: Moisture content difference per slices and group basis ¹

Group 1

(100% LMT)

Group 2

(50% LMT and P)

Group 3

(33.3% LMT, P, B)

Group 4

(25% LMT,P, 50% B)

Group 5

(50% LM T and B)

Cathode

(GM)

17.95 10.02 10.82 9.22 28.24

Middle

(GM)

10.91 9.14 10.98 6.20 13.13

Anode

(GM)

17.11 10.54 13.59 9.97 31.71

¹: In percentage values (%)

GM and B are abbreviations for growth media and biosolids

Figure 3.13: Moisture content difference in EK cell per group basis

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6

Cathode (G.M) Anode (G.M) Middle (G.M) (Group №)

(%)

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3.4.4 Discussion

The EK-GM procedure enabled the strongly bound metals specially the heavy metals to detach

through the introduction of 1 mV∙cm¯¹ of DC field. The process enabled electromigratory,

electrophoretic and different desorption transport reactions to occur throughout the growth media. As

a result effluent was generated that contained the eight metallic elements, especially in the high cation

exchange capacity growth media Groups of 2, 3 and 4. The direct current field enabled the flow of

water towards the cathode. On the other hand peat was assumed to be amphotheric in its nature,

enabling it to change radical valency throughout the experimental procedure.

The application of the DC field on the growth media enhanced the availability of the hard bound

metals like V, Cr, Fe and Li in comparison to the column wash procedure in the generated leachate. The

migration of the subsurface contaminants as a result of different processes like hydrolysis,

electromigration and metal harvesting might be a useful approach for the enhancement of the

phytoextraction and phytostabilization processes by the hyperaccumulant plant. It might also be

applied at certain growth periods of the hyperaccumulant plant. Hence it might increase the overall

efficiency of metal uptake and subsequent physiological storage.

The metal content difference between the EK and Cl was significant. The metal mass content in the EK

leachate was at least three times more than the column leachate. The difference in metal mass

content was assumed to be the result of the direct current of 1 mV∙cm¯¹ application. It initiated

electromigratory and electrophoretic displacements of the hard bound metals. The whole process was

assumed to be due to the disintegration of the lignin coating of the peat ingredient and exposing its

metal content pool. This dissociation enabled the detachment of the hard bound metals from its sub

surface radicals and rendering them detectable in the generated effluent.

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Chapter four: Phyto-sequestration/stabilization

Phase one: Phytoremediation

4.1.1 Introduction

The process of phyto-sequestration (phytoextraction) and phytostabilization evaluation was

accomplished by direct sowing of the Brassica juncea Czern var. Cutlass (accession CN: 46238) seeds.

The five different growth media formulation mixes were placed in a completely random order of

subsamples of four (n=4). After germination the seedlings were grown for eighty six consecutive days.

They were harvested, dried, ashed, and stored. The different physiological parts of the

hyperaccumulant plant were digested in concentrated (12N) hydrochloric acid. Likewise the growth

media surrounding the rhizosphere for the eight metals mass content. The results were summarized in

tables 4.2 and 4.3.

Finally, the efficiency of Brassica juncea hyperaccumulate plant was evaluated based on its efficiency

of removal (E of R), bioaccumulation ratio (BAR), translocation index (TI) and the relative growth rate

(RGR) illustrated in table 4.4.

4.1.2 Sowing plant seeds in different growth media

The seeds of Brassica juncea Czern var. Cutlass (accession CN: 46238) or commonly known as Indian

mustard was brought from “Agriculture and Agri-Food Canada”, research branch in Saskatoon,

Saskatchewan. The seeds were spherical in shape, approximately 1 mm in diameter with pale white in

color. The seeds were sown directly on the five different growth media. They were irrigated once in

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every other day with 10 ml of the organic fertilizer (figure 4.2) amended with 3 g of lithium chloride

diluted in 1 litre of tap water on volume per volume basis (v:v).

After germination the seedlings were grown for eighty six consecutive days. The temperature and

moisture were kept constant as in standard testing and measurement requirements. The lightning

duration was 18 by 6 (18 light and 6 dark) photoperiod hours [249, 250]. The Woods TIM 1000™ light

period regulator was used, (figure 4.1) with four 65 Watts Sylvana spot-grow™ light bulbs. Moreover,

the hyperaccumulant plants were harvested, cleaned, dried, digested and analyzed for their metal

mass content.

4.1.3 Seeding on the five different growth media mixes

The seedlings were grown in pots of 10 cm by 9 cm in size. The five different soil media misses were

arranged in subsamples of four pots (n=4) in a completely random order. Later on they were thinned to

one plant per every pot. [249,106]

The plants were grown for eighty six days. Furthermore, they were irrigated by organic fertilizer once

in two consecutive days. It was composed of 10 ml of the organic fertilizer (figure 4.2) amended with 3

Figure 4.1: Woods TIM 1000™ timer Figure 4.2: Organic fertilizer

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g of lithium chloride diluted in one litre of water on volume per volume basis (v: v). The irrigated

volume for every pot was specific to each growth media’s water holding capacity.

Table 4.1: Void volume and porosity per group basis

Treatment

Group №

LMT, Peat and de-B mixture

( % content )

Void Volume

(ml)

Porosity

(%)

1 100 % LMT 40.27 2.5

2 50 % LMT and 50 % Peat 59.33 3.77

3 33.33 % LMT, Peat and de-B 104.65 6.66

4 25 % LMT, 25 % Peat and 50 % de-B 150.66 9.59

5 50 % LMT and 50 % de-B 116.88 7.44

4.1.4 Harvesting process

The hyperaccumulant plants were harvested after eighty six days of growth (figure 4.3). The lower

and the upper harvestable physiological parts of the hyperaccumulant plants were separated and

visually checked. They were washed and rinsed by tap water for the shortest possible period by a soft

sponge. The washing procedure removed the impurities that were present on its leaves, stem and

especially around its roots. [250, 251]

After the washing process the plants were dried immediately. As a result it stabilized the tissue and

stopped enzymatic reactions to occur [252]. Finally the fresh and the dried weights were recorded of

the different physiological parts of the hyperaccumulant plant. It included its below ground and upper

physiological parts like the leaves, stems, roots which were packed and refrigerated.

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Figure 4.3: Chronology of germination of Brassica juncea in Group 4

→ The Brassica juncea plant seedling

germinated on the first week.

→ The Brassica juncea plant side view.

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Figure 4.3: Chronology of growth of Brassica juncea in Group 4, Cont’d

→ Brassica juncea plant ready to

harvest

→ Aerial view of Brassica juncea plant

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4.1.5 Drying process

The drying process removed the water content that was bound inside the plant tissue. It effectively

stopped the enzymatic reactions that might have led to premature decomposition and sample loss. In

addition water removal facilitated particle size reduction process and homogenization, thus resulted

in a precise and accurate weighting [252].

The pre drying process was achieved after the plant samples were harvested and were placed inside

paper bags. Afterwards, the plant samples were placed in an oven at 80 ⁰C for two consecutive days

[253]. As a note, a temperature below 80 ⁰C wouldn’t have removed the combined water totally from

the tissue sample. Otherwise above 80 ⁰C would have decomposed the plant sample totally. [254]

The dried samples were placed inside sealed bags. It prevented the samples from absorbing moisture

from its surrounding. The dried plant samples were grinded and passed through the 1mm (mesh 20

sieve) screen. Finally the samples were thoroughly mixed and a 5g of aliquot withdrawn for analysis.

The remaining samples were carefully stored and refrigerated.

4.1.6 Storing process

The dried samples were stored in sealed envelopes. It preserved the samples throughout the

experimental duration. In addition, the grinded and homogenized samples were also placed in sealed

envelopes. It preserved its integrity and guaranteed its longevity. Finally the samples were refrigerated

under 4 ⁰C. [253]

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4.1.7 Plant leaf analysis for lithium

Two fully developed leaves were selected randomly. Subsequently, the leaves were washed with

deionized water. The leaves were air dried for one day. Moreover, the leaves were oven dried at 50 ⁰C

for two consecutive days which avoided the volatilization of lithium. The leaves were grinded in a mill

and were passed through a 40 size mesh sieve. In addition a 0.25 g of finely grinded material was

placed in a beaker and was added upon 3 ml of concentrated hydrochloric acid in mass per volume (m:

v) basis. The digestion was allowed to proceed for two consecutive days. Finally the solution was

filtered through Whatman No.1 filter paper of particle retention size of 11 µm. It was transformed to a

25 ml volumetric flask and was bulked by deionized water.

4.1.8 Plant leaf ashing process

The high temperature oxidation method (ashing) was regarded suitable for quantitative analysis of

magnesium, chromium, iron, calcium, sodium, potassium and vanadium. The ashed sample was

digested with concentrated (12N) hydrochloric acid. The filtered sample was aspired by flame atomic

absorption spectrometry (AAS). [255]

Thus a 500 +/- 5 g of the oven dried plant material was weighted and placed into the porcelain

crucible. The crucible was placed in a muffle furnace for 4 hours at 500 ⁰C. A one gram of the ashed

sample was digested in a 10 ml of concentrated (12N) hydrochloric acid for two days. Moreover, the

suspended solution was transferred into a 50 ml volumetric flask and diluted to the volume with

deionized water. Finally the content was analyzed and metal mass content was achieved through the

flame AAS. [252, 254]

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4.1.9 Analysis for the remaining metals in leaf tissue samples

4.1.9.1 Introduction

The Perkin Elmer Analyst 100™ flame AAS was used as stated earlier for the detection of metal mass

contents in different plant physiological parts.

4.1.9.2 Procedure

A 1 g of ashed plant sample was weighted. A 2 ml of concentrated (12N) hydrochloric acid solution

was added and the suspension was left to react for 2 days. Afterwards the suspension was filtered and

diluted with deionized water into a 100 ml volumetric flask. [256]

In plant sciences the concentration of calcium, iron, magnesium, potassium and sodium in plant tissue

solution are expressed as a mass per volume basis. A concentration of 1 to 10 μg/ml of detection limit

0.002 for calcium, 2 to 10 μg/ml of detection limit 0.005 for iron, 0.1 to 0.2 μg/ml of detection limit

0.0003 for magnesium and 1 to 10 μg/ml of detection limit 0.005 for sodium is found usually. However,

chromium and vanadium concentrations in plant tissues range between 0.006 to 18 mg/kg and 0.16 to

1 mg/kg respectively. [258, 254,253,257,259]

4.1.10 Plant stem analysis procedure for extraction of lithium

The stems were separated from the leaves and the roots. They were washed carefully with deionized

water which removed its surface impurities. The stems were left to dry under room conditions for one

day. Afterwards, the samples were oven dried for 12 hours at 50 ⁰C. Furthermore, they were grinded

and passed through a 40 size mesh sieve.

A 0.25 g of the finely grounded material was added upon a 3 ml of concentrated (12N) hydrochloric

acid. The digestion was allowed to continue for two consecutive days. Finally the solution was filtered

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through Whatman No.1 filter paper with particle retention size of 11 µm. The concentrate was

transformed to a 25 ml volumetric flask and was bulked by deionized water.

4.1.11 Analysis for the remaining minerals in plant stem tissue samples

A 1 g of ashed plant stem sample was taken. A 2 ml of concentrated (12N) hydrochloric acid was

added. The concentrate was diluted into a 100 ml volumetric flask [256]. The seven metallic element

concentrations were measured through the flame AAS method as stated earlier.

4.1.12 Plant root analysis procedure for extraction of lithium

The roots were separated from the hyperaccumulant plant stem. Its integrity and unity was preserved

as much as possible. The roots were thoroughly washed and cleaned. They were oven dried for 2

consecutive days at 50 ⁰C. [260]

The roots were grinded in a mill and were sieved through a 40 size mesh size sieve. Furthermore, a

0.25 g of the finely grounded material was placed in a beaker. A 3 ml of concentrated (12N)

hydrochloric acid was added. The digestion was allowed to continue for two consecutive days. Finally

the solution was filtered through Whatman No.1 filter paper of particle retention size 11 µm. The

concentrate was transformed to a 25 ml volumetric flask and was diluted by deionized water. The

lithium metal content concentration was analyzed by the flame AAS.

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4.1.13 Analysis for the remaining metals in the plant root samples

A 1 g of ashed plant stem sample was weighted. A 2 ml of concentrated (12N) hydrochloric acid was

added and the digestion was allowed to proceed for 2 days. The concentrate was diluted into a 100 ml

volumetric flask [256]. Finally its metal mass content was measured through the flame AAS.

4.1.14 Metal concentration analysis in rhizosphere area

A 2 g of oven dried sample was taken. A 20 ml of concentrated hydrochloric (12N) acid was added; in

accordance to ASTM-D3974, 2010. The growth media mass to volume ratio was 1:10. The suspension

was shacked on a BURRELL- Wrist Action™ Pittsburgh, PA USA shaker and was kept to equilibrate

overnight. Furthermore, the samples were centrifuged for 10 minutes at 3,000 rpm and the

supernatant was filtered through Whatman No.1 filter paper of particle retention size 11 µm.

[247,249,246]

Finally the metal mass content was measured through the flame AAS system.

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Table 4.2: Metal mass content in Brassica juncea physiological parts and rhizosphere ¹¯²

Rhizosphere

Mean ± SD

Leaves ²

Mean ± SD

Stem ²

Mean ± SD

Roots ²

Mean ± SD

V 34.168

± 0.370

10.111

± 0.263

18.858

± 0.280

5.090

± 0.206

Li 0.988

± 0.328

0.319

± 0.044

0.084

± 0.005

0.005

± 0.001

Cr 0.513

± 0.090

0.159

± 0.097

0.296

± 0.053

0.065

± 0.050

Fe 1.235

± 1.047

0.142

± 0.658

0.042

± 0.834

0.014

± 0.702

Mg 1.083

± 0.204

0.148

± 0.985

0.306

± 0.052

0.014

± 0.002

Ca 0.855

± 0.046

0.336

± 0.093

0.190

± 0.033

0.031

± 0.013

Na 0.076

± 0.727

0.011

± 0.031

0.031

± 0.089

0.005

± 0.049

K 2.641

± 0.543

1.590

± 2.425

1.555

± 1.255

0.035

± 0.029

¹: In hydrochloric acid matrix

²: Metal mass content in mg∙g¯¹ per dry weight basis

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Figure 4.4: Metal mass content in Brassica juncea physiological parts ¹

¹: Metal mass content in mg∙g¯¹ per dry weight basis

Rhizosphere Leaves

Stem Roots

0

5

10

15

20

25

30

35

V

Li

Cr

Fe

Mg

Ca

Na

K

Rhizosphere

Leaves

Stem

Roots

Metals

mg∙

gr¯¹

pe

r d

ry w

eigh

t b

asis

Plant physiological parts

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Figure 4.5: Li and Cr phytoextraction and phytostabilization in different Brassica juncea physiological

parts ¹

¹: Metal mass content in mg∙g¯¹ per plant dry weight basis

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Rhizosphere

Leaves

Stem

Roots

Li Cr

(mg.g¯¹/Dry Weight )

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Figure 4.6: Vanadium phytoextraction and phytostabilization in different Brassica juncea plant

physiological parts ¹

¹: Metal mass content in mg∙g¯¹ per plant dry weight basis

0

5

10

15

20

25

30

35

Rhizosphere

Leaves

Stem

Roots

V (mg.g¯¹-Dry Weight)

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Table 4.3: Remaining metal mass content, after phytoextraction ¹¯²¯³

Concentration ²

(mean± SD)

Metal

0.513 ± 0.154 Li

1.140 ± 25.859 V

0.276 ± 0.084 Fe

0.352 ± 0.028 Cr

0.333 ± 0.127 Mg

0.342 ± 0.132 Ca

0.447 ± 0.057 Na

0.941 ± 1.520 K

¹: In Group 4 growth media only

²: Metal mass content in mg∙g¯¹, assuming uniformity

³: Cold digestion procedure (12N HCL-matrix)

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Figure 4.7: Comparison between prior and after phytoextraction of metals mass content ¹¯²

¹: Metal mass content in mg∙g¯¹, assuming uniformity

²: In Group 4 growth media

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

Li V

Fe Cr

Mg Ca

Na

K After Before

(mg)

(Metals)

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4.1.15 Mass balance calculation

It was achieved through the computation of the following formula;

C’ = C∙ Vn ∙ (df)/W)}

Where; C’= metal mass content in the sample in mg.kg¯¹

C= measured metal concentration in the solution in mg.l¯¹

Vn= acid volume in l.

W= mass of the initial sample in g.

df = dilution factor.

[261,262]

4.1.16 Effectiveness of different GM mixes

The total metal mass content in the hyperaccumulant plant physiological parts was measured.

Furthermore, it was compared with the remaining metal mass content in each growth media. The

efficiency and percentage removal of the metal mass was calculated in terms of botanical efficiency

parameters (section 4.1.17). [7]

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4.1.17 Parameters of efficiency

Different botanical efficiency parameters were used to assess the phytosequestration and

phytoextraction efficiency of the hyperaccumulant plant. The efficiency of removal was expressed in

percentage value. The botanical efficiency parameters of metal removal were represented in

efficiency of removal, bioaccumulation ratio, the relative growth rate and the translocation index.

→ Efficiency of metal removal (E of R) in (%) = (weight of metal in shoots + weight of metal in roots

(g) /Total metal weight in pot (g) ∙ 100

{Equation: 1}, [249]

→Bioaccumulation ratio of a metal (BAR) = Concentration found in plant tissue / Conc. in soil

i.e. on dry weight basis (DW) {Equation: 2}, [7]

→Relative growth rate (RGR) = {ln(X₂) – ln(X₁)}/ (T₂ - T₁) Where;

X₁ = the height of the plant at time T₁ in (cm) at the start of the experiment.

X₂ = the height of the plant at time T₂ in (cm) at the harvest period of the

experiment.

{Equation: 3}, [263]

→Translocation Index (TI) = weight of metal in plant upper physiology (DW)/ weight of metal in

plant roots (DW)

{Equation: 4}, [264]

i.e per plant dry weight basis (DW)

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Table 4.4: Brassica juncea efficiency parameters ¹

Metal Li Cr V

Efficiency of removal (%)

(E of R)

35 71 161

Bioaccumulation ratio

(BAR)

0.345 0.714 1.612

Translocation index

(TI)

70.931 6.954 5.691

Relative growth rate

(RGR)

0.032

¹: Unit less numbers

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Figure 4.8: Groups 1 and 5 growth media formulations

→The Group 1 growth media

composed of 100% LMT

→The group 5 growth media

composed of 50% LMT and

50% dewatered municipal

biosolids.

i.e. note the formation of the

rigid on top.

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4.1.18 Discussion

The determination of the metal mass content inside the different physiological parts of the

hyperaccumulant plant was the cornerstone of the research. Thus the Brassica juncea was able to

phytoextract and phytostabilize lithium in lieu with chromium and vanadium in a heterogeneous acidic

rhizosphere of pH 4.64 (table 4.4). The outcome paved the way for a possible phytoremediation under

natural circumstances.

The hyperaccumulant plant seeds were planted in the five different growth media formulations of

subsamples of four (n=4). The success of germination, phytoextraction and stabilization was made

possible only in Group 4 pots. It was composed of 25% LMT, 25% homogenized peat and 50%

dewatered municipal biosolids. Its pH was 4.64, oxidation reduction potential was 139.3mV and its EC

was 728 µS∙cm ¯¹ However it was regarded as the suitable formulation under such growth

circumstances.

The other remaining groups utterly failed to sustain growth. Consequently they failed to provide a

suitable growth media for phytosequestration/extraction and phytostabilization processes to occur. As

a result, the hyperaccumulant plant failed to germinate, therefore phyto-harness lithium in lieu with

chromium and vanadium.

It was presumed that the reason behind such an outcome was related to the interaction between the

pH, the ORP (oxidation reduction potential) and the EC of the five different growth media groups. It

tended to influence the Brassica juncea plant germination, growth and maturity stages [293].

According to Ernst [294], heavy metals compete with essential macronutrients in acidic medium. They

sometimes can be taken in excess amounts that might hinder early seed germination and plant growth

periods. As an example in Groups 2 (pH= 3.74, ORP= 197.5 mV and EC= 538 µS∙cm¯¹) and 3 (pH= 4.04,

ORP= 170.7 mV and EC= 893 µS∙cm¯¹). Their pH was below the physiological need of the Brassica

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juncea hyperaccumulant plant (pH between 4.3 and 8.3). The relatively low pH that was recorded in

Group 4 (pH 4.64) prolonged hyperaccumulant plant seed germination time stunted its growth and

extended its phytosequestration/extraction period. As a proof, its the very low relative growth rate

parameter of 0.0325. Moreover, it prolonged the phytoextraction and phytostabilization period of the

hyperaccumulant plant to 86 days. The acidic growth media impacts were clearly observed on the plant

surface. It was characterized by stunted growth, discoloration on the older leaves and premature

senescence. It was also reflected through the low values of its bioaccumulation ratios (BAR), of 0.3456,

0.7142 and 1.6128 for lithium, chromium and vanadium respectively.

According to Belouchi et al. [295], the further acidification of the rhizosphere was a direct result of

root exudates. The process enhanced metal bioavailability and increased its mobility around the plant

root cells. Facilitating its uptake through the secondary pumps on the root cellular level and

translocating it throughout the hyperaccumulant plant physiology. These pumps are made up of

proteins that get triggered by the presence of abundance of H⁺ ions.

In addition, the oxidation reduction potential in Group 4 growth media was 139.3 mV. According to

Belouchi et al. [295], the root plasma membrane potential around the epidermal cell layer might

exceed – 200 mV. This gradient difference generates a strong suction force on both sides.

The difference between the outside and the inner epidermal cell enabled the suction force to occur.

This lead to the subsequent uptake of the heavy metals bound to nitrates or phosphates into its

vascular system. The metals thereafter were transported throughout the xylem system to the upper

physiological parts of the hyperaccumulant plant. These metals were presumably stored as precipitates

in different forms, like carbonates, sulphates and phosphates inside plant vacuoles, Golgi bodies and

endoplasmic reticulum. [295,264,296]

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The translocated index (TI) of lithium, chromium and vanadium was 70.931, 6.954 and 5.6913 (table

4.4) respectively. The closeness of the last two values between the chromium and the vanadium was

presumably due to the closeness of their hydrated Van der Waals (VdW) radii length. Furthermore, the

chemical forms of V (V) and Cr (VI), were suspected to be present in Group 4 growth media. These

forms exist under natural environmental conditions in anionic forms of VO₄¯³ and CrO₄¯². These forms

are readily accessible to plant uptake and subsequent storage.

As for Group 1 (100% LMT), which was regarded as the control, with pH= 7.92, ORP= -33mV and an

EC= 1264 µS∙cm¯¹ and the Group 5 (50% LMT and 50% dewatered biosolids), with a pH= 7.16, ORP= -

5.5mV and an EC= 2103 µS∙cm¯¹. The failure of hyperaccumulant seed germination and subsequent

growth was assumed to be due to the inability of the above mentioned two growth media to hold and

store water sufficiently. This was assumed to be due to the absence of the homogenized peat

ingredient from their formulations. Moreover, a quick visual inspection upon Group 5 (figure 4.8),

showed a formation of a rigid opaque layer on its upper surface which was due to the drying of the

dewatered biosolids with its surrounding lithium mine tailing. The rigid layer formed deprived the seed

from light and water contact.

In Group 1 (figure 4.8-b), the absence of hyperaccumulant seed germination was due to the absence

of water holding capacity. The irrigated water (amended with organic fertilizer and LiCl) was easily lost

due to evaporation and percolation processes.

The first botanical parameter was the efficiency of metal removal (E of R). It was defined as the

percentage of the metal mass in shoots divided by the total metal mass remaining in the growth media

[249]. It was represented by a percentage value. The Brassica juncea’s efficiency of removal for lithium,

chromium and vanadium were 35%, 71% and 161% respectively.

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The second parameter was bioaccumulation ratio (BAR). It was defines as the metal mass content in

the plant tissue divided by the metal mass remaining in the pot. The BAR for lithium, chromium and

vanadium were 0.3456, 0.7142 and 1.6128. It reflected the hyperaccumulant plant ability to

translocate twice amount of vanadium rather than chromium and lithium into its different

physiological parts.

The third criterion was the relative growth rate (RGR) of the hyperaccumulant plant. It showed the

relative growth rate of the hyperaccumulant plant under specific growth circumstances at two

different time and height intervals of the hyperaccumulant plant. The RGR criterion was based on the

growth media characteristics of pH, ORP and EC. The germination and growth of the hyperaccumulant

plant occurred solely in the heterogeneous acidic rhizosphere of Group 4. Therefore, the RGR value

was 0.0325 which was regarded as very low due to the acidic nature of the rhizosphere.

The forth plant efficiency parameter was the translocation index (TI) of the three metals, lithium,

chromium and vanadium in the hyperaccumulant plant physiology. Thus the TI represented a

comparison of metal mass content in upper versus the lower physiology of the hyperaccumulant plant.

In the continuous phytoremediation process the higher the value of translocation index, the more

successful the plant was regarded. This is because the precipitated metals in the shoots are more easily

harvested, recycled or stored as opposed to its roots. As a result, the translocation index for lithium

was 70.931, for chromium 6.954 and for vanadium 5.6913. Therefore, the hyperaccumulant plant was

extremely successful in translocating lithium, rather than chromium and vanadium and precipitating it

inside its upper physiology.

On the rhizosphere level of Group 4, it was assumed that due to the abundant content of dewatered

municipal biosolids (50% of the total growth medium weight) was successful in improving the metal

uptake specially lithium from the growth medium. However, it was speculated that the different

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organisms like mycorrhizal fungi, strains of Bacillus and pseudomonas formed symbiotic relationships

with the Brassica juncea hyperaccumulant plant roots. [264]

Moreover, these root colonizing bacterial organisms might have played a pivotal role in the

sequestration and mobilization of these metals to the hyperaccumulant plant roots system [1].

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Phase two: Monocotyledonous plant phytoextraction and phytostabilization analysis

4.2.1 Introduction

The monocotyledonous (grass) plant belongs to the Poaceae or Gramineae families. It encloses more

than eleven thousand species and over 800 genera in total, mostly annuals but few perennials. The

grasses are essential food producers like rice (Oryza sativa L.), wheat (Triticum aestivum L.), corn (Zea

mays L.), barley (Hordeus vulgare L.) and many others. [267]

Physiologically, the grasses possess fibrous, abundant and very dense root system. They are regarded

as suitable for phytoextraction and phytostabilization of soil pollutants. Their leaves are linear with

parallel veins vertically pointed. The grasses are extremely successful in tolerance to various

ecosystems due to their varied means of reproduction and photosynthesis. Moreover; grasses produce

seeds through cross pollination, self fertilization, and asexual reproduction through vegetative parts

like rhizomes and stolons. [268]

Finally the aim of planting the monocotyledonous plant seeds in the most favourable growth media

(Group 4) was to quantify its efficiency to phytoextract/stabilize under acidic heterogeneous

rhizosphere and compared it with the dicotyledonous plant (Brassica juncea) results.

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Figure 4.9: Monocotyledonous plant growth and harvest

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4.2.2 Experimental process and results

The monocotyledonous plant seeds were directly sown on the most successful growth media Group 4

of subsamples of four (n= 4). They were left to grow for eighty six days. They were irrigated once every

two days by a 10 ml of the organic fertilizer, amended with 3 g of lithium chloride on weight per

volume basis (w/v) diluted in a liter of water. The irrigated volume of water mixed fertilizer was specific

to its one pore volume.

The hyperaccumulant plants were harvested, dried, stored and its different physiological parts were

digested in concentrated (12N) hydrochloric acid matrix. The metal mass content of lithium and

chromium was analyzed through the Perkin Elmer Analyst 100 ™ flame AAS.

Finally the excess samples were properly stored and refrigerated in caped containers.

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Table 4.5: Metal mass content in monocotyledonous plant ¹¯²¯³

Element Rhizosphere

mean ± SD

Roots

mean ± SD

Leaves

mean ± SD

Remaining GM

mean ± SD

Cr 0.275 ± 0.038 0.319 ± 0.063 0.342 ± 0.092 0.266 ± 0.046

V <LLD <LLD <LLD <LLD

Na 0.256 ± 0.057 1.014 ± 0.684 0.741 ± 0.073 0.313 ± 0.129

K 0.779 ± 0.451 1.721 ± 2.872 8.094 ± 1.682 0.807 ± 0.988

Li 0.228 ± 0.037 0.592 ± 0.058 2.211 ± 0.644 0.551 ± 0.032

Fe 0.456 ± 0.439 0.535 ± 0.326 0.433 ± 0.125 0.456 ± 0.148

Ca 2.536 ± 0.084 0.946 ± 0.228 0.250 ± 0.074 1.263 ± 0.109

Mg 0.427 ± 0.093 0.877 ± 0.065 1.197 ± 0.408 0.589 ± 0.321

¹: Concentrations in mg∙g¯¹ per dry weight basis

²: <LLD as low level of detection

³: The GM as the growth media

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Table 4.6: Monocotyledonous plant efficiency parameters ¹

Metal

Cr

Li

Efficiency of removal (%)

(E of R)

122

360

Bioaccumulation ratio

(BAR)

1.22

3.6

Translocation index

(TI)

1.071

3.730

Relative growth rate

(RGR)

0.052

¹: Unit less numbers

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Figure 4.10: Li and Cr phytoextraction and phytostabilization in monocotyledonous plant ¹

¹: Metal mass content in mg∙g¯¹ per plant dry weight basis

0

0.5

1

1.5

2

2.5

Leaves

Roots Rhizosphere

Cr Li

(mg∙g¯¹/Dry Weight basis)

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Figure 4.11: Brassica juncea vs. monocotyledonous plant phytoaccumulation of Li and Cr ¹ ¹ Harvestable weights constituted the added leaf and the stem metal masses

0

0.5

1

1.5

2

2.5

MC (Li) Brassica juncea (Li) MC (Cr) Brassica juncea (Cr)

Rhizosphere Root Harvestable

mg∙

g¯¹ p

er d

ry w

eigh

t b

asis

.

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Figure 4.12: Brassica juncea vs. monocotyledonous plant in botanical efficiency terms for Li and Cr ¹

¹ Mono: The abbreviation for the monocotyledonous plant

0

50

100

150

200

250

300

350

400

Mono. (Cr) Brassica

juncea (Cr) Mono (Li) Brassica

juncea (Li)

Bioaccumulation ratio Relative growth rate Translocation index Efficiency of removal (%)

(Par

amet

ers

)

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4.2.3 Discussion

The aim of sowing the monocotyledonous plant seeds, directly on the most favorable growth media

was to compare it, with the dicotyledonous plant the Brassica juncea. It was sown under homogeneous

growth conditions. It was compared with the dicotyledonous plant based on the botany wise efficiency

parameters (tables 4.4 and 4.6).

The monocotyledonous surpassed the Brassica juncea in efficiency of removal, relative growth rate,

and bioaccumulation ratios but lagged behind in translocation index. The monocotyledonous plant was

more than two times successful in its relative growth rate. Moreover, it was twice more efficient in

removal and in bioaccumulation ratios for chromium and more than ten times for lithium, rather than

the Brassica juncea hyperaccumulant plant.

On the other hand the Brassica juncea was six times more efficient in translocating chromium and

more than twenty times for lithium inside its upper harvestable physiological parts rather than the

monocotyledonous plants.

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Chapter five: Conclusion

This research showed the capability of Brassica juncea to phytoharness 0.408 mg∙g¯¹ of lithium in its

different physiological parts per dry weight basis. Also it phytostabilized 0.988 mg inside its

heterogeneous acidic of Group 4.

Brassica juncea’s efficiency of lithium in lieu with chromium and vanadium of removal were 35%,

71% and 161%, but with an extremely low relative growth rate of 0.0325.

Brassica juncea bioaccumulation ratio for lithium, chromium and vanadium were 0.345, 0.714 and

1.61 and their translocation indexes were 71, 7 and 5.6 respectively.

Five different growth media groups were formulated and tested. The most favourable one was the

Group 4 (pH 4.64, ORP 139.3 mV, EC 728 µS∙cm¯¹ and TDS 436.8 mg∙l¯¹). It supported

hyperaccumulant plant seed germination, growth and permitted phytostabilization and

phytosequestration/extraction to occur.

Macronutrient presence throughout the five different groups ranged between 0.142 mg to 0.589

mg, which were regarded as very low but were supplemented through the mixed water and organic

fertilizer irrigations.

Monocotyledonous plant was able to phytoextract 2.803 mg∙g¯¹ and 0.561 mg∙g¯¹ of lithium and

chromium per its dry weight basis. Similarly it phytostabilized 0.228 mg and 0.275 mg of lithium and

chromium inside its heterogeneous acidic rhizosphere.

The comparison between the two hyperaccumulant plants; the Brassica juncea and the

monocotyledonous were achieved through the botany wise efficiency parameters. The relative

growth rate of the monocotyledonous plant was twice that of the dicot plant. Moreover, the

efficiency of removal and bioaccumulation ratios was more than twice and ten times that for

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149

chromium and lithium. On the contrary the Brassica juncea’s translocation index was more than six

and twenty times for chromium and lithium, compared with the monocotyledonous plant.

A novel proposal for an efficient phytosequestration/stabilization and proper reclamation for mine

tailing. It included the incorporation of ingredients, such as peat and dewatered municipal biosolids

incorporated with the tailing mass. However, for optimal phytoharnessing results an integrated

mixed planting pattern between the monocot and the dicot hyperaccumulating plants might be a

viable alternative. Maximizing the overall process efficiency might include the coupling of an EK field

at the vigorous growth stage of the hyperaccumulant plants. In addition, the engineered ecosystem

might mimic the role of a natural buffer and a reservoir minimizing the metal mass runoffs in the

lithosphere.

Future work recommendations

Determination of lithium allelopathic symptoms and effects on plant growth and reproduction.

Determination different lithium precipitation forms and sink sites on the hyperaccumulant plant

cellular level.

An insitu trial of the novel integrated approach for mine tailing reclamation, phytosequestration

/stabilization under natural circumstances.

Assessing new techniques like; heat treatment, electrorefining and other conventional metal

recovery procedures for the recovery of the phytomined metals specially lithium.

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150

Appendix

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Table 5.1: LMT texture data in detail ¹¯²

Mesh

Sieve № (mm)

Empty Weight

(g)

Final Weight

(g)

Remaining Soil

(g)

Percentage

Retained

(%)

Percentage

Passed

(%)

4 (4.76 mm) 490.37 493.78 3.41 0.341 99.65

8 (2.36 mm) 497.41 506.61 9.2 0.920 98.739

10 (2.00 mm) 438.10 458.80 20.7 2.070 96.669

12 (1.68 mm) 493.39 1201.45 708.06 70.814 25.855

16 (1.19 mm) 442.39 624.65 182.26 18.228 7.627

30 (600 μm) 442.63 511.84 69.21 6.922 0.705

50 (247 μm) 361.02 363.15 2.13 0.213 0.492

100 (150 μm) 360.48 362.75 2.27 0.227 0.265

200 (75 μm) 342.70 343.96 1.26 0.126 0.139

Collecting Pen 369.80 371.19 1.39 0.139

Total 999.89 100

¹: The size of sand was divided into three types coarse; that retained on sieve № 10, medium on sieve

№ 30 and finally fine retained on sieve № 200.

²: SD ± 0.1 mg.

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